ES2530091T3 - Peptide compositions for the treatment of cancer by inhibiting the activity of the calcium channel TRPV6 - Google Patents

Abstract

Isolated peptide comprising: a) an amino acid sequence with a sequence identity of at least 90% with the amino acid sequence ** Formula ** or ** Formula ** or b) at least 11 amino acids of the amino acid sequence: wherein said peptide inhibits calcium channel activity without paralyzing activity.

Description

Peptide compositions for the treatment of cancer by inhibiting the activity of the calcium channel TRPV6

Field of the Invention

The invention relates to peptides for use in the reduction of cell proliferation and the treatment of cancer, including metastatic cancer.

Background of the invention

A 54 amino acid paralytic peptide called soricidin (NCBI accession number P0C2C6) was discovered and isolated from the submaxillary salivary gland of the northern colicorta shrew (Blarina brevicauda). An earlier patent on the use of soricidine to treat pain (U.S. Patent No. 7,119,168 disclosed data

10 demonstrating that this 54-residue peptide caused paralysis and inhibited calcium uptake in two ovarian cancer cell lines (U.S. Patent No. 7,273,850). Antineoplastic drugs that have no paralyzing activity are still needed.

A group of calcium ion channels involved in cancer are the transient receptor potential (TRP) channels found in invertebrates and vertebrates. A member of the TRP superfamily received its name after it was discovered that it was activated in the presence of vanilloids (the capsaicin of hot peppers, for example) and was called transient receptor potential for vanilloids. The first four receptors that were found to be of this type (TRPV1, TRPV2, TRPV3 and TRPV4) responded to capsaicin and were also responsible for detecting temperature changes. The other two of the TRPV subfamily, TRPV5 and TRPV6, were predominantly found in epithelial tissues and were responsible for the entry of calcium ions. It was identified that TRPV5 / 6 was responsible for the importation of calcium into the epithelial tissues of the intestine and, therefore, for the uptake of calcium from the diet. It was also shown that these channels are present in a number of different tissues in different amounts, but in more quantity in the intestinal epithelial cells, kidney, placenta and pancreas. TRPV6 expression was measured and was very high in human ovarian, prostate and breast cancers, thyroid and colon tumors, and in some cell lines

25 known prostate cancer. In prostate cancer, TRPV6 was highly induced in carcinomas that had crossed the tissue wall and had begun to metastasize (Zhuang et al., 2002). Therefore, TRPV6 is a possible target for cancer treatment. Consequently, there is an enormous need for compounds that block the activity of the overabundant TRPV6 channels in cancer cells.

More recently it has been suggested that the involvement of TRPV6 in cancer activates a pathway

30 survival / antiapoptotic in two cancer cell lines with overexpression of TRPV6. In the prostate cancer cell line (LnCaP), the intracellular calcium concentration, increased by the large amount of TRPV6, activates the nuclear factor of activated T lymphocytes (NFAT; Lehen'kyi et al. 2007). Bolanz et al. (2008) have also demonstrated the implication of calcium-activated NFAT in changing antiapoptotic genes in a human breast cancer cell line (T 47-D). Bolanz et al. showed that production reduction

35 of TRPV6 by interfering RNAs seemed to relieve apoptotic blockade. These results further accentuate the need for TRPV6 inhibitors for cancer treatment.

Compendium of the invention

The inventors have synthesized isolated peptides that have calcium channel inhibitory activity and, in particular, calcium channel inhibitory activity of TRPV6. Peptides are useful for treating cancer, including metastatic cancer. Surprisingly, these compounds have a sequence identity with a continuous chain of amino acids of soricidine, although they have the calcium channel inhibitory activity without the paralyzing activity. There was no evidence that the structure of soricidine that had calcium channel inhibitory activity was separated from the structure that caused the paralyzing activity. The peptides of the invention are, optionally, half the length of the soricidine, or are smaller. The peptides of the invention also

45 have unexpectedly more calcium channel inhibitory activity than soricidin in some cells. It was also determined that inhibitory activity increased in some cells as the length of the peptides decreased.

The peptides of the invention have other unexpected properties compared to full-length soricidine, such as greater solubility, longer shelf life and lower antigenic capacity. In one embodiment, the invention 50 is refers to an isolated peptide comprising all or part of the amino acid sequence

(SEQ ID No. 1), wherein the peptide inhibits the activity of the channel

calcium and lacks paralyzing activity. The peptide is optionally

o. Another aspect relates to the peptides of the invention for use in a method of inhibiting calcium uptake in a cancer cell, inducing cell apoptosis and / or preventing or treating cancer, which comprises administering to the

Cell all or part of a peptide of the invention, wherein the peptide inhibits calcium uptake in the cell

cancerous The cancer optionally includes breast cancer, ovarian cancer, blood count, brain cancer, retinal cancer, liver cancer, thyroid cancer, colon cancer, prostate cancer or endometrial cancer. The peptides of the invention inhibit the activity of the calcium channel without paralyzing activity, such as the activity of the calcium channel of TRPV6. The cancer optionally comprises a metastatic cancer, such as a

5 metastatic cancer located in a lymph node, lung tissue, kidney tissue or liver tissue.

Another embodiment of the invention relates to a pharmaceutical composition comprising a peptide of the invention and a carrier. The invention includes peptides of the invention to be used in the preparation of a medicament for inhibiting calcium channels in a cell, inducing apoptosis of a cancer cell and / or treating cancer. The invention also includes peptides of the invention to be used in the inhibition of

10 calcium channels of a cell, the induction of apoptosis of a cancer cell and / or the treatment of cancer.

Other features and advantages of the present invention will be apparent from the following detailed description. However, it should be known that the detailed description and specific examples, although indicating preferred embodiments of the invention, are only offered by way of illustration, since from the detailed description different changes will be apparent to those skilled in the art. modifications within the spirit and

15 of the scope of the invention.

Brief description of the drawings

Embodiments of the invention will be described in relation to the drawings, in which:

Figure 1 shows the average net change in calcium current (ISOC) through the TRPV6 channels.

depending on the concentration of peptide (panel A). SorC13 is represented by hollow circles and a line

20 discontinuous. SorC27 is represented by hollow squares and a continuous line. Note that the positive numbers indicate the inhibition (net current) of the flow of calcium current into the cell. Panel B shows the percentage of maximum calcium current inhibition (ISOC) through the TRPV6 channels as a function of the peptide concentration. SorC13 is represented by hollow circles and dashed line. SorC27 is represented by hollow squares and a continuous line.

25 Figure 2 shows the effect of SorC27 on an ovarian cancer cell line (SKOV-3, panel A) and a breast cancer cell line (MCF 7, panel B).

Figure 3 shows the effect of SorC13 on an ovarian cancer cell line (SKOV-3, panel A) and a

breast cancer cell line (T 47D, panel B).

A comparison of the induction of apoptosis by paclitaxel (10 µM) and SorC13 is shown in Figure 4

30 (10 µM) in the T 47D (panel A). Panel B shows a comparison of paclitaxel (10 µM) and SorC27 (10 µM) in MCF 7.

Figure 5 shows a comparison of the proportion of treatment with CAT: control of CAT (without CAT) with the proportion of treatment with SorC13: control of Sorc13 (without SorC13) and treatment with SorC27: control of SorC27 (without SorC27) on the SKOV3 (panel A). Panel B shows the comparison of treatments with CAT and

35 with SorC13 on CaOV3. The concentration of SorC13 was 100 µM; that of SorC27 was 10 µM. CAT = paclitaxel (10 µM) plus carboplatin (20 mM). The data of each point corresponds to the average of four measurements of the induction of apoptosis. The dotted line is a 1: 1 ratio obtained for the absence of effect.

Figure 6 shows a comparison of the proportion of treatment with CAT: control of CAT (without CAT) with the

Proportion of treatment with SorC27: control of SorC27 (without SorC27) over OVCAR3. The concentrations of

40 SorC27 were 10 µM (filled square) and 100 µM (empty square). CAT = paclitaxel (10 µM) plus carboplatin (20 mM). The data of each point corresponds to the average of four measurements of the induction of apoptosis. The dotted line is a 1: 1 ratio obtained for the absence of effect.

Figure 7 is a linear drawing showing the location of lymph nodes in the mouse. Accumulation

significant of SorC13-Cy5.5 and SorC27-Cy5.5 four hours after injection i.v. 100 µg of each of

45 labeled peptides in CD1 mice were observed in the following lymph nodes marked in Figure 7: 1, superfacial cervical nodes; 4, axillary ganglia; 5, gill nodes; 8, mesenteric ganglia; and 9, inguinal nodes.

The distribution of Cy5.5 labeled SorC13 in CD1 mice is shown in Figure 8, 4 hours after

i.v. The Y axis is the percentage of the total fluorescence measured in all tissues.

50 Figure 9 shows the distribution of Cy5.5 labeled SorC27 in CD1 mice, 4 hours after i.v. The Y axis is the total fluorescence measured in each tissue.

The distribution of Cy5.5 labeled SorC27 in CD1 mice after injection is shown in Figure 10

i.v. over time after infusion to remove fluids. The Y axis is the percentage of fluorescence

Total measure in all tissues. The highest percentage of (total fluorescence) uptake of SorC27 was observed in liver, lung and kidney. The lymph node is not shown because the infusion removes the lymph. Panel A shows the distribution 4 hours after the injection i. v and in panel B, 24 hours after the injection i.v.

Figure 11 shows the degradation of SorC27 in human blood plasma from HPLC 5 analysis of 100 µl of plasma diluted in 100 µl of 2M KCl, 94 µl injection. The Y axis indicates the amount (µg) of SorC27 in the plasma before dilution with KCl, half-life = 20.0 min, r = 0.87, p <0.05. Data are mean ± SEM, n = 6.

The results of an in vivo experiment with NOD / SCID mice xenografted with human SKOV3 ovarian cancer tumors are shown in Figure 12. The mice were injected with control saline, SorC27 or a mixture of paclitaxel and carboplatin (CAT). SorC27 significantly reduced tumor volume (p <0.05)

10 when compared with an injection of control saline and did not differ from mice treated with CAT for 12 days when tumor size was normalized by body mass.

Detailed description of the invention

The inventors have manufactured isolated peptides that have calcium channel inhibitory activity without paralyzing activity. In particular, the peptides have TRPV6 calcium channel inhibitory activity and block the

15 TRPV6 function in cancer cells that have overexpressed TRPV6. Peptides are useful in cancer treatment procedures, such as breast cancer, ovarian cancer, blood count (leukemia), brain cancer, retinal cancer, liver cancer, thyroid cancer, colon cancer, prostate cancer and endometrial cancer.

Peptides are also useful for the treatment of metastatic cancer that has originated from a first tissue and that extends to a secondary site, such as a lymph node, liver, lung or kidney tissue.

In one embodiment, the peptide of the invention has the amino acid sequence:

(called "SorC9"; amino acids No. 19-27 of SEQ ID No. 1). In another embodiment, the compound has the sequence of amino acids

(called "SorC13"; amino acids No. 15-27 of SEQ ID No. 1). In another embodiment, the compound comprises all or part of the amino acid sequence.

(called "SorC27"; SEQ ID No. 1).

The invention also describes an isolated nucleic acid encoding a peptide of the invention, such as a nucleic acid encoding SEQ ID No. 1 or one of its fragments described herein. The invention also describes antibodies isolated against a peptide of the invention. The antibody optionally and selectively binds to a peptide of the invention, but does not bind to soricidine.

Surprisingly, the compounds of the invention have sequence identity with part of the soricidine, but have calcium channel inhibitory activity without paralyzing activity. Previously it was not known that soricidine had two functional domains in its structure. It was also not known that peptides that separated the calcium channel inhibitory activity from the paralyzing activity could be prepared. The peptides of the invention are optionally half the length of the soricidine or are smaller. The peptides of the invention not only

35 isolate and preserve the calcium channel inhibitory activity, but unexpectedly have a higher calcium channel inhibitory activity than soricidine in certain cell lines, such as OV 2008. In some cell lines the inhibition activity was also increased. as the length of the peptide decreased.

The surprising nature of this invention is accentuated by considering that, while large and bifunctional enzymes and proteins are prevalent in biological systems, inherent bifunctionality is a very rare phenomenon in small peptides. Published results on bifunctionality have typically been the result of artificial production, for example, when two different peptides have been chemically fused (Anes et al. 2006; Yamamoto et al. 2008). The inventor determined that the amino end of soricidine has a paralyzing function and that the carboxyl end has a calcium channel inhibitory function. The truncation of soricidin by the amino terminus successfully produced peptides that conserved the inhibitory activity of the

45 calcium without paralyzing activity.

The peptides of the invention have other unexpected properties compared to full length soricidine. For example, the peptides of the invention are more soluble, which allows smaller doses to be administered to achieve a desired peptide concentration in the blood plasma. Based on the relative solubilities, the dose volume of the peptides of the invention to achieve a concentration

50 desired blood could be at least 10 times smaller than that of soricidine.

Additionally, the peptides have greater storage and solution stability due to the presence of less sensitive disulfide bridges (the soricidine has 3 disulfide bridges). The peptides of the invention do not optionally have disulfide bridges. An absence of disulfide bridges provides a very long storage period when preserved with minimal cooling or at room temperature as a solid.

Additionally, when the peptides of the invention without disulfide bridges dissolve in water, the disulfide bridges cannot be cleaved. Such cleavage, in full-length soricidine, can make the peptide less active and more prone to form intermolecular disulfide bridges. The formation of interpeptide disulfide bridges also results in the precipitation of soricidin. Peptides of the invention, such as SorC13

5 and Sorc27, are typically stable in aqueous solution at 8 ° C for at least 3 weeks without any change in purity, as measured by HPLC.

The peptides of the invention also dodge one of the main adverse effects of pharmaceutical substances related to the ability to cross the protective barrier of the central nervous system: the blood brain barrier. That the peptides of the invention are unable to cross this protective barrier

10 eliminates possible toxicity in the central nervous system.

The peptides of the invention have advantageously little toxicity. As shown in example 23, after the i.v. In vivo in CD1 mice, SorC27 did not change blood pressure or heart rate during a measurement period of 1 hour. Nor was any neurological / behavioral change observed for 72 hours. After the injection i.v. In vivo in the CD1 mice, SorC13 caused a small peak 15 (approximately 25%) of blood pressure in the first 15 min that disappeared after 1 hour. No neurological / behavioral changes were observed in the mice for 72 hours. Single intravenous injection of SorC13 or SorC27 in CD1 mice at doses of 10 mg / kg, 100 mg / kg and 500 mg / kg produced no adverse events during an observation period after the 5-day injection. The autopsy also showed no significant changes in the main organ systems. In addition, a multiple dose of SorC27 at 400 mg / kg (i.p.) each

20 days for 12 days in the mice showed no indication of toxicity.

Peptides of the invention, in particular smaller peptides, such as SorC13, typically have less antigenic capacity than soricidine. Peptides that have an amino acid number equal to or less than the empirical threshold for antigenicity (typically considered to be 13 amino acids for peptides in general) do not possess antigenic ability.

As noted above, a peptide of the invention will have calcium channel activity (ie, the ability to partially or totally inhibit channel activity) without paralyzing activity. Peptides having such activity are readily identified with any suitable known assay that measures i) calcium channel blockage and ii) the absence of paralyzing activity. For example, in one embodiment, a peptide having calcium channel inhibitory activity is optionally identified by determining that the peptide reduces channel activity.

30 calcium by reducing (namely, partially or totally inhibiting) the flow of calcium through calcium channels.

The calcium channel inhibitory activity is optionally identified by an easily available cell line (e.g., human embryonic kidney cell lines, HEK, lymph node prostate cancer cell, LnCaP) transfected with an expression vector for TRPV6 . Such transfected cells are easily distributed in aliquots and preserved (typically at -80 ° C) until needed. This provides a preparation of standard transfected cells to test the inhibition of calcium ion uptake in the cells. The analysis of the movement of calcium in the cell lines to identify the peptides of the invention that provide a reduction in calcium channel activity is optionally performed by fluorometric measurements (internalized FURA calcium ion probe) or by an electrophysiological protocol that involves the zonal clamping of the cells and measuring the calcium current in the presence or absence of a peptide. 40 Optionally, the peptides have an equilibrium inhibition constant of less than: 1000 nM, 150 nM or 100 nM in a model of LnCaP cells at the same time as they do not alter the transmission by the sciatic nerve of an action potential in a model of rat sciatic nerve (namely, without paralyzing activity). For example, the equilibrium dissociation constant (Kd) for SorC27 is 140 nM and that of SorC13 is 100 nM in the LnCaP model (Figure 6). Based on a linear relationship with the number of amino acids, the Kd of SorC9 is expected to

45 is approximately 90 nM.

Isolated peptides are useful for a number of purposes. One embodiment includes peptides of the invention for use in methods of reducing cell proliferation by administering a peptide of the invention to a cell, such as a cancer cell. The reduction of cell proliferation is readily determined by contacting a peptide with a proliferating cell, such as a cancer cell, in vitro or in vivo, and

50 determine whether the peptide reduces (namely, completely or partially inhibits) cell proliferation. In particular, the invention describes methods of inducing apoptosis of a cell, such as a cancer cell, by administering a peptide of the invention to the cell. The reduction of cell proliferation is readily determined by contacting a peptide with a cell, such as a cancer cell, in vitro or in vivo, and determining whether the peptide induces apoptosis.

55 As indicated above, isolated peptides are useful for treating cancer. The invention describes methods for treating cancer in a mammal, such as a human, by administering an isolated peptide of the invention to the mammal. Notably, in the independent direct and comparative trials against the well-known cancer drug, paclitaxel, and the cancer drug cocktail, paclitaxel and carboplatin ("CAT cocktail"), the two peptides of the invention, SorC13 and SorC27 , provided one more induction

rapid and more active apoptosis in certain cell lines. This effect has been shown to be better against both breast cancer and ovarian cancer. A typical treatment against breast cancer is paclitaxel and a typical treatment against ovarian cancers is the CAT cocktail. In other cell lines, the operation of SorC13 and SorC27 was comparable to that of CAT.

5 It has also been shown that the SorC27 isolated peptide is useful for treating cancer in a mouse model with in vivo xenograft. As shown in Example 14 and Figure 12, in xenografted NOD / SCID mice with human ovarian cancer tumors, SorC27 significantly reduced tumor volume (p <0.05) when compared to the situation of control (saline injection) and did not differ from mice treated with CAT, for 12 days, when tumor size was normalized by body mass.

10 Without intending to compromise with the theory of the cause of the antineoplastic effect, the inventor has determined, through experiments with SorC27 and SorC13, that the peptides of the invention activate the apoptotic cascade through caspase 3 and / or 7 in ten cell lines different cancerous, but not in two non-cancerous cell lines.

Thus, a peptide of the invention is optionally identified as having activity against cancer if it causes

15 induction of the enzymes caspase 3 and caspase 7 above the levels of caspase 3 and caspase 7 in the control cells. Once these enzymes are activated, cell death will occur. The activity of caspasse enzymes in the treated cells is easily measured and compared with the untreated control cells.

In addition, as shown in Table 9 and in Example 25, the presence of TRPV6 mRNA in a cell line corresponds to cell lines that show an apoptotic effect in response to the peptides of the

20 present invention.

Metastatic cancer

The peptides of the invention have antineoplastic activity against a cancer cell at a primary tumor site or a cancer cell that metastasizes at a secondary cancer site. Peptides are predominantly located in the lymph nodes, but also in the lung, liver and kidney, which are the usual sites

25 in which metastatic cancer is located (Figure 7).

The peptides of the present application are useful for the treatment of metastatic cancer, including a cancer that has spread to the lung, brain, liver, kidney, spleen and bone marrow. The peptides can be used alone or in a pharmaceutical composition comprising a second cancer drug.

The peptides of the present application are particularly useful for treating lymph node metastases.

30 "Lymph node metastasis" optionally includes lung cancer (Mujoomdar et al., 2007), gastric cancer, cervical carcinoma (Lyshchik et al., 2007), vulvar carcinoma (Vernooij et al., 2007), endometrial cancer (Aalders et al., 2007), squamous cell carcinoma of the head and neck (Veness et al., 2007), esophageal and throat cancer, nasopharyngeal carcinoma (Ma et al, 2007), digestive cancer (Wind et al., 2006), gallbladder cancer, brain cancer (Mujoomdar et al., 2007), thyroid cancer, breast cancer, ovarian cancer, cancer

35 prostate and colorectal cancer. Thus, the peptides of the invention are useful for treating cancer in a mammal at any stage of cancer I, II, III or IV.

Peptides of the invention

Isolated peptides that comprise all or any part of SEQ ID No. 1 and that have calcium channel inhibitory activity (TRPV6 inhibitory activity), without paralyzing activity, are peptides useful for the treatment of cancer. Amino acids can be added to the peptides of the invention, but the isolated peptides, with amino acids, are typically 35 or 30 amino acids or less, optionally less than: 27, 25, 20, 15 or 13 amino acids in length, even optionally at least 9 amino acids in length. Longer peptides can be easily made by adding a series of additional amino acids to the SorC27 sequence to make a peptide that could be, for example, up to 30, 35, 40 or 45 amino acids in length (e.g. , the amino acids

45 which correspond to as one or more of the amino acids of the carboxyl end (SEQ ID No. 2), a targeting sequence or other amino acids).

The peptide optionally comprises, consists essentially of or consists of the amino acid sequence:

minus: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of SEQ ID No. 1. Optionally, the isolated peptide comprises at least: 9, 10, 11, 15 or 18 amino acids of SEQ ID No. 1. Optionally, the peptide comprises a fragment of 9-13, 10-15, 15-20, 20-25 or 20-27 amino acids of SEQ ID No. 1, where the peptide inhibits calcium channels, reduces cell proliferation and has no paralyzing activity.

The peptides of the invention also optionally include analogs of the above-mentioned peptides. The analogs of the protein of the invention optionally include, but are not limited to, an amino acid sequence that contains substitutions, insertions, deletions and / or mutations of one or more amino acids. The amino acid substitutions may be of a conservative or non-conservative nature. Conservative amino acid substitutions involve replacing one or more amino acids of the peptides of the invention with similar loading, size and / or hydrophobicity amino acids. When only conservative substitutions are made, the resulting analog must be functionally equivalent. Non-conservative substitutions involve replacing one or more amino acids of the amino acid sequence with one or more amino acids that have a different load, size and / or hydrophobicity characteristics. The analog is optionally a peptoid,

10 which is an N-substituted polyglycine with R amino acid groups attached to the N atom.

One or more amino acid insertions are optionally introduced into the amino acid sequences of the invention. The amino acid insertions consist of a single amino acid residue or sequential amino acids that range, for example, from 2 to 15 amino acids in length.

Deletions consist of the removal of one or more amino acids, or isolated portions of the 15 amino acid sequence from the peptide. The amino acids removed may be contiguous or they may not be.

The peptides of the invention are readily prepared by chemical synthesis with techniques well known in protein chemistry, such as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85: 2149-2154)

or synthesis in homogenic solution (Houbenweil, 1987, Methods of Organic Chemistry, ed. E. Wansch, vol. 15 I and II, Thieme, Stuttgart).

The analogs of a protein of the invention are optionally prepared by introducing mutations into a nucleotide sequence encoding the peptide. Mutations in the nucleotide sequences constructed for the expression of analogs of a protein of the invention retain the reading frame of the coding sequences. In addition, the mutations will preferably not create complementarity regions that could hybridize to produce secondary structures in the mRNA, such as loops or hairpins, that could affect

25 adversely to the translation of mRNA.

Mutations are optionally introduced into particular locuses by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites that allow ligation to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes an analog having the desired insertion, substitution or deletion of amino acids.

Alternatively, oligonucleotide-directed site-specific mutagenesis procedures are employed to provide an altered gene that has certain altered codons in accordance with the required substitution, deletion or insertion. Deletion or truncation of a peptide of the invention is also easily achieved using appropriate restriction endonuclease sites adjacent to the desired deletion. After the restriction, the protuberant ends can be filled in and the DNA religed. The sample procedures for manufacturing

35 alterations presented above are described by Sambrook et al. (Sambrook J et al., 2000. Molecular Cloning: A Laboratory Manual (third edition), Cold Spring Harbor Laboratory Press).

In addition, analogs of a protein of the invention are readily prepared by chemical synthesis with techniques well known in protein chemistry, such as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85: 2149-2154) or the synthesis in homogeneous solution (Houbenweyl, 1987, Methods of Organic 40 Chemistry, ed. E. Wansch, vol. 15 I and II, Thieme, Stuttgart). Peptides of the invention also include peptides having a sequence identity with a peptide of the invention, mutated peptides and / or truncation thereof, as described herein. Such peptides have amino acid sequences that correspond to nucleic acid sequences that hybridize under stringent hybridization conditions (see discussion of stringent hybridization conditions herein) with a probe used to obtain

A peptide of the invention. Peptides that have the same sequence will often have regions that are characteristic of the protein.

Other useful peptides of the invention optionally comprise, consist essentially of or consist of an amino acid sequence with at least: a sequence identity of 80%, 90% or 95% with all or part of SEQ ID No. 1 described in The present specification, wherein the peptide has calcium channel inhibitory activity and no paralyzing activity, and is useful for the treatment of cancer. Sequence identity is typically assessed by advanced search with the BLAST version 2.1 program (parameters like above; Altschul, SF, Gish, W., Miller, W., Myers, EW and Lipman, D. J (1990) « Basic local alignment search tool. ”J. Mol. Biol. 215: 403-410). BLAST is a series of programs that are available online through the US National Center. UU. for Biotechnology Information (building 38A of the National Library of Medicine, Bethesda, MD 55 20894). Advanced search with BLAST is adjusted with the default parameters. References for BLAST programs include: Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. (1990) "Basic local alignment search tool". J. Mol. Biol. 215: 403-410; Gish, W. and States, D. J. (1993) "Identification of protein coding regions by database similarity search". Nature Genet 3: 266-272; Madden, T. L., Tatusov, R. L. and Zhang, J. (1996)

Applications of network BLAST server Meth. Enzymol 266: 131-141; Altschul, SF, Madden, TL, Schäffer, AA, Zhang, J., Zhang, Z., Miller, W. and Lipman, DJ (1997) «Gapped BLAST and PSI-BLAST: a new generation of protein database search programs» . Nucleic Acids Res. 25: 3389-3402); Zhang, J. and Madden, T. L. (1997) "PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation." Genome Res. 7:

5 649-656).

The present invention also describes a protein of the invention conjugated with a selected protein or

a selectable marker protein to produce fusion proteins.

Therapeutic procedures

Peptides of the invention, such as all or part of SEQ ID No. 1 described herein, are useful.

10 to reduce cell proliferation, induce cell apoptosis and prevent or treat cancer by administering the peptide to a human.

The phrase "reduce cell proliferation" as used herein refers to slowing the proliferation rate of a cell compared to the proliferation rate of a cell in the absence of the substance.

15 The phrase "induce cell apoptosis", as used herein, refers to increasing the speed of cell apoptosis compared to the speed of cell apoptosis in the absence of the substance.

The term "effective amount" as used herein means an effective amount and in doses and for the periods of time necessary to achieve the desired result (eg, optionally,

20 block calcium channel activity, reduce cell proliferation, induce apoptosis and / or prevent or treat cancer).

Administering a peptide or substance to a mammal includes administrations both in vivo and ex vivo.

The terminology "a cell", as used herein, includes a single cell as well as a plurality or population of cells. Administration of a peptide or a substance to a cell includes

25 administrations both in vitro and in vivo.

The phrase "reduces the activity of the calcium channel", as used herein, means that the substance may lead to a decrease in the activity of the calcium channel compared to the activity of the calcium channel in the absence. of the substance

The peptides of the invention strongly inhibit calcium uptake in cancer cells, in particular the

30 cancer cells in which the calcium uptake channel of TRPV6 has increased expression, such as in breast cancer, ovarian cancer, blood count, brain cancer, retinal cancer, liver cancer, thyroid cancer, cancer colon, prostate cancer and endometrial cancer. The peptides of the invention, by reducing calcium uptake, alter the intracellular calcium essential for the proliferation of normal and cancer cells. Accordingly, the invention includes the use of a peptide of the invention to reduce proliferation.

35 induce apoptosis and / or prevent or treat tumors and cancer in mammals (eg, humans) by administering the peptide to the mammal.

As used herein, and as is well known in the art, "treating" or "treatment" is a strategy for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical outcomes may include, but are not limited to, the relief or improvement of one or more.

40 symptoms or conditions, decrease in the extent of the disease, stabilized state (namely, that does not worsen) of the disease or disorder, prevention of the spread of the disease or disorder, delay or slowing of the progression of the disease or disorder , improvement or palliation of the state of the disease or disorder, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to expected survival if no treatment is received.

45 Pharmaceutical compositions

The invention also includes the use of the peptides of the invention to prepare a medicament for the treatment of cancer. The isolated peptides of the invention are optionally formulated in a pharmaceutical composition for administration to subjects in a biologically compatible form suitable for in vivo administration. With "biologically compatible form suitable for in vivo administration" is intended

50 refer to a form of the substance to be administered in which any toxic effect has less weight than therapeutic effects. The substances can be administered to living organisms, including humans and animals.

The administration of a therapeutically active amount of the pharmaceutical compositions herein

invention is defined as an effective amount, in a few doses and for periods of time necessary to

Get the desired result. For example, a therapeutically active amount of a substance may vary according to factors such as the disease status, age, gender and weight of the individual, and the ability of the substance to trigger a desired response in the individual. . Dosage dosages can be adjusted to provide the optimal therapeutic response. For example, they can be administered

5 several independent doses per day or the dose can be reduced proportionally as indicated by the requirements of the therapeutic situation.

The peptide of the invention is preferably combined with other components, such as a vehicle in a composition, such as a pharmaceutical composition. The compositions are useful when administered for drug treatment or cancer prevention.

The pharmaceutical compositions can be administered to humans or animals by a series of procedures, including but not limited to topical administration, oral administration, aerosol administration, intratracheal instillation, intraperitoneal injection, injection into cerebrospinal fluid, intravenous injection and subcutaneous injection. The doses to be administered depend on the patient's needs, the desired effect and the route of administration chosen. Nucleic acid molecules and

The peptides can be introduced into cells with in vivo administration vehicles such as liposomes. They can also be introduced into these cells by physical techniques such as microinjection and electroporation, or chemical procedures such as coprecipitation, pegylation or by liposomes.

The pharmaceutical compositions are prepared by known methods for the preparation of pharmaceutically acceptable compositions that can be administered to patients, and such that an effective amount of the nucleic acid molecule or peptide is combined in a mixture with a pharmaceutically acceptable carrier. . Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA) or Handbook of Pharmaceutical Additivies (compiled by Michael and Irene Ash, Gower Publishing Limited, Aldershot , England (1995)). On this basis, the compositions include, but not exclusively, solutions of the substances in

Association with one or more pharmaceutically acceptable carriers or diluents and may be contained in buffered solutions with a suitable pH and / or be isoosmotic with physiological liquids. In this regard, reference can be made to US Pat. UU. No. 5,843,456.

On this basis, pharmaceutical compositions optionally include an active compound or substance, such as a peptide or nucleic acid molecule, in association with one or more carriers or diluents.

30 pharmaceutically acceptable, and contained in buffered solutions with a suitable pH and isoosmotic with physiological liquids. Procedures for combining active molecules with vehicles or combining them with diluents are well known to those skilled in the art. The composition optionally includes a selective action agent for transporting the active compound to the specified sites within the tissue.

Antibody preparation

35 Antibodies against the peptide are useful for identifying the presence of the peptide in a test sample. Any procedure to label the antibody that reports the density / location of the peptide would be useful.

(e.g., radioactively labeled peptide or fluorescently labeled peptide). The antibody is typically a monoclonal antibody or a polyclonal antibody. Antibodies are also valuable for immunopurification of peptides. For example, a biological sample can be contacted with the antibody under the conditions that allow the formation of an immune complex between the antibody and a peptide recognized by the antibody, and detect in the sample the presence or absence of the immune complex whereby the presence of the peptide of the invention is detected. The invention also describes compositions that preferably include the antibody, a suitable means for the formation of an immune complex between the antibody and a peptide recognized by the antibody, and a reagent capable of detecting the immune complex to check the

Presence of the peptides of the invention.

To recognize the peptides of the invention, antibodies can be generated against a range of unique epitopes along the peptides.

Monoclonal and polyclonal antibodies are prepared according to the description in this application and the methods known in the art. For examples of preparation procedures and uses of monoclonal antibodies, see US Pat. UU. Nos. 5,688,681, 5,688,657, 5,683,693, 5,667,781, 5,665,356, 5,591,628, 5,510,241, 5,503,987, 5,501,988, 5,500,345 and 5,496,705. Examples of the preparation and uses of polyclonal antibodies are described in US Pat. UU. 5,512,282, 4,828,985, 5,225,331 and

5,124,147.

The term "antibody", as used herein, includes fragments thereof that

They also react specifically with a peptide of the invention. The antibodies can be fragmented with conventional techniques and the usefulness of the fragments can be counted in the same manner as described above. For example, F (ab ') 2 fragments can be generated by treating the antibody with pepsin. The F (ab ') 2 fragment

The resulting can be treated to reduce disulfide bridges to produce Fab 'fragments.

The invention also describes methods of using antibodies, such as for the detection of receptors that bind to the peptides of the invention. For example, the invention includes a method for detecting the presence of a peptide of the invention, wherein: a) a sample containing one is contacted

5 or more peptides with an antibody of the invention under the appropriate conditions for which the antibody binding is given to the peptides with which it reacts specifically; b) unbound peptides are separated from the antibody; and c) the antibody that remains bound to one or more of the peptides in the sample is detected.

Research tool

The peptides of the invention are useful in search protocols to explore neuromuscular junction and

10 ion channels. The ability to selectively alter certain ion channels or classes of ion channels provides another tool with which to alter the neuromuscular synapse in a predictable manner. This identifies the function of sensitive peptide targets in neuromuscular functions and processes. The invention includes a method for determining the response of an ionic channel to a paralyzing peptide, which comprises contacting a channel or cells comprising a channel with a peptide of the invention or a derivative thereof and

15 determine whether the channels carry ions or if the ion transport has been reduced (eg, Ca2 +).

The peptides of the invention inhibit TRPV6 channels and are easily labeled with a label (fluorescent labeling, radioactive labeling, biotin labeling, etc.) identifying the TRPV6 channel in a cell or tissue, or cell marking or tissues that express a large amount of calcium channels. Accordingly, the invention relates to methods for identifying a TRPV6 channel in a cell or tissue,

20 which comprises contacting the cell or tissue with a peptide of the invention that has been labeled with a detectable label and detecting the peptide bound to the TRPV6 channel in the cell or tissue.

Nucleic acids

The peptides of the invention (including truncations, analogs, etc.) can be prepared by chemical synthesis or by recombinant DNA methods. Accordingly, the invention describes nucleic acid molecules having a sequence encoding a peptide of the invention. These sequences are readily incorporated in accordance with the procedures known in the art in a suitable expression vector that ensures good peptide expression. Expression vectors include, but are not limited to, modified cosmids, plasmids or viruses (eg, retroviruses, adenoviruses and adeno-associated viruses with defective replication), as long as the vector is compatible with the host cell used. The expression "suitable vectors

30 for the transformation of a host cell 'means that the expression vectors contain a nucleic acid molecule of the invention and regulatory sequences, selected based on the host cells to be used for expression, which are operably linked to the nucleic acid molecule .

"Operatively linked" means that the nucleic acid is linked to regulatory sequences in a manner that

allows the expression of nucleic acid.

Thus, the invention describes a recombinant expression vector of the invention containing a nucleic acid molecule of the invention, or a fragment thereof, and the regulatory sequences necessary for transcription and translation of the inserted peptide sequence. Suitable regulatory sequences optionally come from different sources, including bacterial, fungal or viral genes (for example, see the regulatory sequences described in Goeddel, Gene Expression Technology: Methods in Enzymology 185,

40 Academic Press, San Diego, Calif. (1990)). The selection of suitable regulatory sequences depends on the host cell chosen and can be easily carried out by one skilled in the art. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or binding sequence for an RNA polymerase, a ribosomal binding sequence, which includes a translation initiation signal. Additionally, depending on the host cell chosen and the vector used, other sequences can be incorporated into the expression vector,

45 such as an origin of replication, additional DNA restriction sites, enhancers and sequences that confer transcription inducibility. It can also be appreciated that the necessary regulatory sequences can be provided by the native compound and / or its flanking regions.

The invention further describes a recombinant expression vector comprising a nucleic acid molecule.

of DNA of the invention cloned into the expression vector in an antisense orientation. These vectors are

50 experimental systems useful for studying the peptides of the invention or its variants, or for testing antidotes. Peptides may be toxic, or they may not be, to host cells. They are also useful for producing large amounts of the peptide.

The recombinant expression vectors described herein may also contain a selection marker gene that facilitates the selection of transformed or transfected host cells with a recombinant molecule of the invention. Examples of selection marker genes are genes that encode a protein such as G418 and hygromycin that confer resistance to certain drugs, β-galactosidase,

Chloramphenicol acetyltransferase or firefly luciferase. Transcription of the selection marker is followed by changes in the concentration of the selection marker protein, such as β-galactosidase, chloramphenicol acetyltransferase or firefly luciferase. If the selection marker gene encodes a protein that confers antibiotic resistance, such as neomycin resistance, the transforming cells can be

5 select with G418. The cells that have incorporated the marker selection gene will survive, while the other cells will die. This makes it possible to visualize and test the expression of recombinant expression vectors of the invention and in particular to determine the effect of a mutation on the expression and the phenotype. It will be appreciated that selection markers can be introduced into a separate vector from the nucleic acid of interest.

Recombinant expression vectors may also contain genes encoding a fusion part that

10 increases the expression of the recombinant protein; increases the solubility of the recombinant protein; and helps purify a desired recombinant protein by acting as a ligand in affinity purification. For example, a proteolytic cleavage site can be added to the desired recombinant protein so that the recombinant protein can be separated from the fusion moiety after purification of the fusion protein.

Recombinant expression vectors can be introduced into host cells to produce a cell.

15 transformed host. These cells are useful experimental systems. Accordingly, the invention describes a host cell comprising a recombinant expression vector. The term "transformed host cell" is intended to include prokaryotic and eukaryotic cells that have been transformed or transfected with a recombinant expression vector of the invention. Prokaryotic cells can be transformed with nucleic acid by, for example, electroporation or calcium chloride mediated transformation. Acid

Nucleic may be introduced into mammalian cells by conventional techniques such as coprecipitation with calcium phosphate or with calcium chloride, transfection mediated with DEAE-dextran, lipofectin, electroporation or microinjection. Suitable procedures for transforming and transfecting host cells can be found in Sambrook et al (Molecular Cloning: A Laboratory Manual (third edition), Cold Spring Harbor Laboratory Press) and others, such as laboratory textbooks.

Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the peptides of the invention can be expressed in bacterial cells such as E. coli, Pseudomonas, Bacillus subtillus, insect cells (via baculovirus), yeast cells or mammalian cells. Other suitable host cells can be found in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1991).

As an example, to produce peptides recombinantly, for example, E. coli can be used with the T7 RNA polymerase / promoter system using two plasmids or by labeling the proteins encoded by the plasmid, or by expression by phage infection M13 mGPI-2. You can also use vectors from

E. coli with phage regulatory sequences λ, by vectors with fusion protein (e.g., lacZ and trpE), by fusions to maltose binding proteins and by glutathione-S-transferase fusion proteins.

Alternatively, a peptide can be expressed in insect cells by baculovirus vectors, or in mammalian cells by variolovaccine virus. For expression in mammalian cells, the cDNA sequence can be linked to heterologous promoters and introduced into cells, such as COS cells, to achieve transient or long-term expression. Stable integration of the chimeric gene construct can be maintained in mammalian cells by biochemical selection, such as neomycin and acid.

40 mycophenolic.

The DNA sequence can be altered with procedures such as restriction enzyme digestion, filling with DNA polymerase, exonuclease deletion, extension by terminal deoxynucleotide transferase, ligation of synthetic or cloned DNA sequences, alteration of the site-specific sequence with the use of specific oligonucleotides together with the PCR. For example, they can be removed or mutated from one to five or from

45 five to ten amino acids or more.

The DNA sequence or portions thereof, or a mini gene consisting of a DNA with an intron and its own promoter, is introduced into eukaryotic expression vectors by conventional techniques. These vectors allow DNA transcription in eukaryotic cells by providing regulatory sequences that initiate and enhance DNA transcription, and ensure their proper support and polyadenylation. The endogenous gene promoter can also be used. Different promoters within vectors have different activities that alter the level of cDNA expression. In addition, some promoters can also modulate functioning, such as the promoter that responds to glucocorticoids from the mouse breast tumor virus.

Some of the vectors listed contain selection markers or neo-bacterial genes that allow the isolation of cells by chemical selection. Vectors can be stable in the long term in the

55 cells as episomic entities that replicate freely through virus regulatory elements. Cell lines can also be produced that have the vector integrated into the genomic DNA. In this way, the gene product is produced continuously.

The vectors are introduced into the target cells by different procedures, including calcium phosphate, strontium phosphate, electroporation, lipofection, DEAE-dextran, microinjection or by fusion of protoplasts. Another alternative is that the cDNA can be introduced by infection with viral vectors.

The peptides of the invention are easily isolated from cells or tissues, including mammalian cells or tissues, in which the peptide is expressed.

The peptide is easily purified by conventional purification procedures known to those skilled in the art, such as chromatography procedures, high performance liquid chromatography procedures or precipitation.

For example, an antiseptic antibody (as described herein) is readily used to isolate a peptide that is then purified by standard procedures.

Isolated peptides of the invention are also readily prepared by chemical synthesis with techniques well known in protein chemistry, such as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85: 2149- 2154) or the synthesis in homogeneous solution (Houbenwely, 1987, Methods of Organic Chemistry, de.

E. Wansch, vol. 15 I and II, Thieme, Stuttgart).

15 Examples

The following examples illustrate embodiments of the invention and do not limit the scope of the invention.

Example 1: Separation of the amino acid sequence of the soricidine calcium channel inhibitor

The amino acid sequences of the carboxyl end region of soricidine were synthesized. These segments have been called SorC13 (which has a sequence identical to that of the 13 amino acids of the carboxyl end of

20 soricidine) and SorC27 (which has a sequence identical to that of the 27 amino acids of the carboxyl end of soricidine). The amino acid sequence of SorC13 and SorC27, as well as some of their physical and physiological properties, are described in Table 1. The half-lives of SorC13 and SorC27 (as shown in example 22) were smaller than the half-life of Sor54.

Table 1: Summary of the comparison of SorC54, SorC27 and SorC13

Property

SorC54 SorC27 SorC13

No. of amino acids

54 27 13

Molecular mass

5806 (3 disulfide bridges) 2957 (no disulfide) bridge 1566 (no disulfide bridge)

Sequence

NCBI Access Number P0C2C6 (SEQ ID No. 1)

pl

4.3 (measured) -5.6 (calculated) -8.3 (calculated)

Physical appearance

White solid White solid White solid

Solubility (aqueous)

<20 mg / ml > 200 mg / ml > 200 mg / ml

Purity

≥ 95% ≥ 95% ≥ 95%

Stability in aqueous solution at 20 ° C

Stable Very stable Very stable

Stability of the solid at 20 ° C

Stable Very stable Very stable

Stability of the solid at room temperature

Unstable Very stable Very stable

Property

SorC54 SorC27 SorC13

Direct HPLC detection limit

~ 200 ng ~ 200 ng ~ 200 ng

Physiology

Elimination route in mice with i.v. 5 mg / kg

Determined Rapid by the kidney Rapid by the kidney

Cellular Diana

Ionotropic sodium channels and TRPV6 TRPV6 TRPV6

Physiological effects in vivo (flour worm); 5 mg / kg

paralyzing Non-paralyzing Non-paralyzing

Physiological effects in vivo

Not rehearsed None observed Peak of 15% TA at

(mice) at 5 mg / kg

for 72 hours 15 min, stabilizes in 1 h, no effect observed in 72 h

Rat plasma half-life

> 30 h; binds to plasma proteins 21 min; does not bind to plasma proteins 58 min; does not bind to plasma proteins

Half-life in human plasma

> 30 h; binds to plasma proteins 20 min; does not bind to plasma proteins 56.6 min; does not bind to plasma proteins

Maximum dose tolerated in CD-1 mice by a single dose i.v., with doses of 10, 100 and 500 mg / kg

Not rehearsed No toxic effect No toxic effect

Repeat the dose in

Not rehearsed No loss of mass No loss of mass

NOD / SCID mice to

body compared body compared

400 mg / g, injection i.p.

with the controls, I don't know with the controls, I don't know

daily for 12 days

observe toxic effects observe toxic effects

"Very stable" in the table above means that there was no observable degradation of the solid peptides for 6 months. As a solution in sterile water, the peptides are typically stable for at least 3 weeks at 8 ° C and at –20 ° C they are expected to be stable for more than 1 year.

5 SorC27 and SorC13 do not show paralyzing activity. To demonstrate this, the paralytic flour worm bioassay described above was used (U.S. Patent No. 7,119,168). Animals from a mealworm colony were arbitrarily selected and placed in groups (treatment and control). Animals were weighed to allow calculation of equivalent doses. The treatment was with SorC13 or SorC27 dissolved in an Ringer solution buffered for insects. The injection control was of an equivalent volume of

10 Ringer's solution for insects (NaCl; 10.40 g; KCl, 0.32 g; CaCl2, 0.48 g; NaHCO3, 0.32 g; dissolved in 1 l of sterile Millipore water, pH 7.4) . The peptide formulations were: SorC27 solutions, prepared at 1.0 mg / 100 µl of Ringer's insect solution (10 µg / µl, 3.4 µM, MM = 2957); and SorC13, prepared at 0.5 mg / 100 µl (5 µg / µl, 3.4 µM, MM = 1566). The doses were 50 µg of SorC27 per 100 mg of the animal mass and 25 µg of SorC13 per 100 mg of animal mass. Peptide solutions and control saline were injected dorsally into the

15 fourth segment from the head, just below the tegument. The animals were gently pinched the tail with forceps to trigger the reaction of the escape reflex and were scored by the time for the effect, the duration of the effect and the intensity of the effect. None of the animals treated with SorC13 or SorC27 showed no remarkable effect, while the animals treated with soricidine showed a deep paralysis.

Table 2 shows the comparison of the number of paralyzed larvae by injecting the C peptides into

comparison with soricidine and control saline injection. The shrew peptide (soricidine) is deeply paralyzing in the flour worm model. When the SorC27 and Sorc13 peptides were injected into the larvae at equivalent molar doses (20 nmol / 100 mg larva), there was no evidence of paralysis.

Table 2. Comparison of the paralytic activity of the shrew soricidine with respect to SorC13 and SorC27 5 from it. The animals were dosed with 20 nmol / 100 mg of mass.

Time (min)

Control (saline solution) Soricidin SorC13 SorC27

0

0/4 4/4 0/4 0/4

2

0/4 4/4 0/4 0/4

30

0/4 4/4 0/4 0/4

Example 2: SorC13 and Sorc27 strongly inhibit calcium ion uptake through the transient receptor potential (by vanilloids) six (TRPV6)

An expression vector (pCAGS-IRES-hTRPV6b) (Bodding et al., 2003) that contains the nucleotide code for the

10 Human TRPV6b was transfected into lymph node rectal cancer cells (LnCaP, ATCC CRL1740) and expressed in them. The zonal clamp with current measurement of the entire cell was used to measure the calcium current through this channel. The cells were treated and the current operated by the calcium reserve (ISOC) was measured with SorC13 (n = 90) or with SorC27 (n = 30) in a closed circuit perfusion for 3 min at each accumulated concentration of 0 ( baseline), 100 nM, 500 nM, 1 µM, 30 µM and 100 µM. Wash infusion was done at 2

15 ml / min for 3 min. The positive control used was MRS-1845, a known reserve-operated channel (SOC) inhibitor (N-propargilnitrendipene; available from Sigma Aldrich) at 5 µM and, in this system, demonstrated the inhibition of calcium current. A summary of the effects of SorC13 and SorC27 is shown in Figure 1. It is clear that both C peptides strongly inhibited the flow of calcium through the TRPV6 channel. The adjustment of the data to a hyperbolic function allowed to calculate the concentration that provides 50% inhibition of SOC, for SorC13

20 (ISOC-50 = 0.10 µM) and SorC27 (ISOC-50 = 0.14 µM). In addition, both C-peptides strongly inhibit the calcium current through the TRPV6 channel, with inhibition constants in the 100 nM range.

Example 3: SorC13 and SorC27 induce apoptosis in human breast cancer and ovarian cancer cell lines, in particular in cancer cell lines with TRPV6

The peptides of the invention are useful for inducing apoptosis in cells in vitro and in vivo, in particular in

25 cancer cells, such as the cell lines listed in Table 3. The cell lines used as a model for human breast cancer and human ovarian cancer were obtained from the American Type Culture Collection (ATCC) and grown in the recommended conditions and in the culture media recommended by the ATCC.

Cancer cell line culture

30 Cell lines were grown under sterile conditions at 37 ° C, in a humidified atmosphere of 5% CO2. The culture media used are collected below for each cell line:

T 47D was cultured in RPMI medium (Sigma-Aldrich) modified with 15% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mg / ml bovine insulin and a penicillin mixture plus streptomycin (at 50 µg / ml each).

MCF-7, MDA-MB-231, MDA-MB-415, MDA-MB-468 were grown in DMEM modified with 10% fetal bovine serum (v / v), 2 mM L-glutamine, and streptomycin and penicillin at 50 µg / ml each.

MCF-10A, MCF-12A were cultured in 50% DMEM plus 50% Ham F12 modified with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mg / ml bovine insulin, 500 hydrocortisone ng / ml, and 50 µg / ml penicillin and streptomycin. The combined medium was supplemented with 5% fetal bovine serum (v / v).

OVCAR-3 was cultured in RPMI 1640 medium supplemented with 1.0 mM sodium pyruvate, 0.01 mg / ml insulin and 10% fetal bovine serum (v / v).

SKOV-3 was grown in McCoy% A medium supplemented with 1.5 mM L-glutamine, 2.2 g / l sodium bicarbonate and 10% fetal bovine serum (v / v).

CAOV-3 was grown in Dulbecco's modified Eagle's medium (glucose at 4.5 g / l) with 4 mM L-glutamine, 1.5 g / sodium bicarbonate and 10% fetal bovine serum (v / v ).

OV-90 was grown in a 1: 1 mixture of MCDB 105 medium and 199 medium supplemented with 15% fetal bovine serum (v / v).

5 HeyC2 was grown in RPMI 1640 medium supplemented with 1.0 mM sodium pyruvate, 0.01 mg / ml insulin and 10% fetal bovine serum (v / v).

Table 3: List of human ovarian cancer and breast cancer cell lines in which the induction of apoptosis was analyzed by SorC13 and / or SorC27.

Cellphone line

ATCC number Description

Human breast cancer lines

MCF 7

HTB-22 Non-invasive epithelial adenocarcinoma of the mammary gland

MDA-MB-231

HTB-26 Invasive mammary epithelial adenocarcinoma

MDA-MB-415

HTB-128 Adenocarcinoma of the human mammary gland

MDA-MB-468

HTB-132 Adenocarcinoma of the human mammary gland

T 47D

HTP-133 Canalicular carcinoma of the human mammary gland

Non-cancerous human breast cell lines

MCF-10A

CRL-10317 Non-neoplastic mammary gland epithelium

MCF-12A

CRL-10782 Human mammary gland epithelium, spontaneous immortalization

Human ovarian cancer cell lines

CaOV-3

HTB-75 Human ovarian adenocarcinoma

OVCAR-3

HTB-161 Human ovarian epithelial adenocarcinoma

OV-90

CRL-11732 Malignant papillary serous adenocarcinoma of human ovary

SKOV-3

HTB-77 Human ovarian adenocarcinoma

HEY C-2

Human ovarian epithelial cancer

10 Lines MCF-7, MDA-MB-468, MCF-10A, MCF-12A and OVCAR-3 were tested for induction of apoptosis with SorC13 and SorC27. The induction of apoptosis with SorC13 was analyzed for lines T 47D and CaOV-3. Lines MDA-MB-231 and MDA-MB-415 were analyzed for induction of apoptosis with SorC27. The other cell lines were analyzed with SorC13 and SorC27 with a similar methodology to demonstrate that C peptides induce apoptosis.

Follow-up of induction of apoptosis and cell viability

Apoptotic induction levels and cell viability were determined by multiplexing the measurements for single samples. The Promega CellTiter Blue and APO-ONE assay systems allow the determination of cell viability correlated with the number of cells, and the induction of apoptosis. This protocol uses a 5-fluorogenic peptide that contains a peptide recognition sequence for caspase 3 and caspase 7, which initiates the proteolytic enzymes of the apoptosis cascade. The CellTiter Blue reagent was added to the wells in a 96-well plate containing the cells to be analyzed, immediately after adding the peptide solution or the vehicle. About 5000 cells were seeded in the wells the day before. At the chosen time (day 1, day 2, etc.), the fluorescence was measured at 590 nm (emission) after excitation at 560 nm. Be

10 measured the activity of caspases 3 and 7 (excitation at 485 nm / emission at 527 nm) in the same wells after adding 120 µl of Apo-ONE reagent, freeze lysing at –80 ° C for 1 h and incubation for 1 h temperature ambient.

For this work, the concentrations of SorC13 and SorC27 used were 0 µM, 1 µM, 10 µM and 100 µM. The experiments covered a period of 5 to 7 days. All measurements were performed in quadruplicate and

15 were used to correct the test measurements both a blank (without cells) and a control (vehicle, without peptide). Response data as a function of time and response as a function of dose were plotted as means ± the standard error of the mean. All statistical comparisons were made with the Student's t-test or by ANOVA analysis during the course of the dose. The level of statistical significance was considered to be the 95% confidence interval (ie, P ≤ 0.05).

20 In cell viability, the induction of apoptosis and the accompanying decrease in breast cancer and ovarian cancer cell lines were observed. The effects of SorC27 on an ovarian cancer (SKOV-3) and breast cancer (MCF 7) cell line are shown in Figure 2. Similarly, the effect of SorC13 on ovarian cancer cell lines and of breast cancer is shown in Figure 3. From the large increase in activity of caspases 3 and 7, it is clear that in the induction of apoptosis there is an effect

25 time dependent and a dose dependent effect.

The observation of the effects of SorC13 on breast cancer cell lines is summarized in Table 4 and shows that:

 SorC13 induced apoptosis and decreased cell viability in three of the four cancer cell lines with a statistically significant value.

30  SorC13 induced T 47D apoptosis (Table 4, Figure 3B) with a statistically significant value.

 SorC13 did not induce apoptosis in the two controls of non-cancerous cell lines (MCA-10A and MCA12A).

The observation of these effects of SorC13 on ovarian cancer cell lines is summarized in Table 5 and shows that:

35  Treatment of 3 ovarian cancer cell lines induced the apoptotic cascade significantly above the 'no treatment' control (positive: CaOV3, OVCAR3, SKOV3).

Table 4: The effect of SorC13 on the induction of apoptosis in human breast cancer cell lines. An apotosic index is shown in the table (AI = response to treatment / response to control; AI = 1 when there is no

40 effect). An indication of whether cell viability decreased (+) or not (-) is also shown. Two non-cancerous breast cell lines were included for comparison (MCF 10A and 12A).

Breast cancer cell line

SorC13 concentration (µM) from Apoptotic index no effect) (1 = Statistical significance Decrease in cell viability

MB 468

one 2.7 P <0.05 +

10

3.3 P <0.05 +

100

2.7 P <0.05 +

T 47D

one 2.0 P <0.05 +

Breast cancer cell line

SorC13 concentration (µM) from Apoptotic index no effect) (1 = Statistical significance Decrease in cell viability

10

2.8 P <0.05 +

100

4.3 P <0.05 +

MCF 7

one 1.2 P> 0.05 -

10

1.1 P> 0.05 -

100

2.4 P <0.001 +

MCF 10A

100 1.0 - -

MCF 12A

100 1.0 - -

Table 5: The effect of SorC13 on the induction of apoptosis in human ovarian cancer cell lines. An apoptotic index is shown in the table (AI = response to treatment / response to control; AI = 1 when there is no effect). An indication of whether cell viability decreased (+) or not (-) is also shown.

Ovarian cancer cell line

SorC13 concentration (µM) from Apoptotic index no effect) (1 = Statistical significance Decrease in cell viability

CaOV3

Day 5

100 2.7 P <0.05 +

OVCAR3

Day 4

100 1.4 P> 0.05

Day 5

100 1.7 P <0.05 +

SKOV3

Day 4

100 2.2 P> 0.05

Day 5

100 2.7 P <0.05 +

The observation of the effects of SorC27 on breast cancer cell lines is summarized in Table 6 and shows that:

 4 breast cancer cancer cell lines treated with SorC27 showed a significant apoptotic response in response to exposure to SorC27 (positive: MB 416, MB 468, MB 231, MCF 7).

 Non-cancerous breast cell lines (MCA 10A and MCA 12A) were not affected by SorC27.

Observations of the effects of SorC27 on ovarian cancer cell lines are summarized in Table 7 5 and show that:

 4 ovarian cancer cell lines (OV90, OVCAR3, SKOV3, HEYC2) induced apoptosis after treatment with SorC27. The effects ranged from 1.9 times to 6.3 times greater than the control condition without any treatment.

Table 6: The effect of SorC27 on a series of human breast cancer cell lines. The table shows a

10 apoptotic index (AI = response to treatment / response to control; AI = 1 when there is no effect). An indication of whether cell viability decreased (+) or not (-) is also shown. Two non-cancerous breast cell lines are included for comparison (MCF 10A and 12A).

Breast adenocarcinoma cell line

SorC27 concentration (µM) Apoptotic index (1 = no effect) Statistical significance Decrease in cell viability

MB 231

+

Day 3

10 1.5 P <0.05

100

1.7 P <0.01

MCF 7

+

Day 2

10 1.0 P> 0.05

100

2.8 P <0.001

Day 3

10 2.9 P <0.05

100

4.6 P <0.001

MB 415

10 1.3 P <0.05 +

100

1.5 P <0.01

MB 468

+

Day 3

10 1.1 P = 0.07

100

1.2 P <0.01

MCF 10A

100 1.0 - -

MCF 12A

100 1.0 - -

Table 7: The effect of SorC27 on a series of human ovarian cancer cell lines. The table shows an apoptotic index (AI = response to treatment / response to control; AI = 1 when there is no effect). An indication of whether cell viability decreased (+) or not (-) is also shown.

Ovarian cancer cell line

from SorC27 concentration (µM) from Apoptotic index (1 = no effect) Statistical significance Decrease in cell viability

HEY C2

+

Day 6

100 µM 1.4 P <0.05

Day 7

10 µM 2.2 P <0.05

100 µM

2.8 P <0.05

OV90

+

Day 2

100 µM 1.8 P = 0.003

Day 3

1 µM 1.2 P = 0.026

10 µM

1.9 P = 0.001

100 µM

1.9 P = 0.001

OVCAR3

+

Day 3

10 µM 2.2 P = 0.0026

100 µM

2.2 P = 0.003

Day 5

10 µM 4.1 P <0.0001

100 µM

6.3 P <0.0001

SKOV3

+

Day 2

10 µM 2.7 P = 0.0008

100 µM

1.5 P = 0.09

Day 3

1 µM 1.7 P = 0.0012

10 µM

2.4 P <0.0001

100 µM

2.7 P <0.0001

Example 4: Analysis of SorC13 and SorC27 against paclitaxel when inducing apoptosis in metastatic human breast cancer cell lines

5 To assess the effect of the two type C peptides by comparison with the reference treatment currently used against breast cancer (paclitaxel), SorC13, SorC27 and paclitaxel (10 µM) were compared. The APO-ONE (Promega) and CellTiter Blue assays were used with the breast cancer cell line panel. Data were compared directly in terms of mean response and analyzed for a significant difference.

10 The cell lines used were T 47D and MCF7, which were obtained from the American Type Culture Collection (ATCC) and were grown under the recommended conditions and in the culture media recommended by the 19

ATCC. Cell lines were grown under sterile conditions at 37 ° C, in a humidified atmosphere of 5% CO2, with the culture medium discussed above.

SorC peptides had a profound effect when compared to paclitaxel. As illustrated in Figure 4, SorC13 was, in all cases, more effective in inducing a faster and more intense apoptotic response and, taking this as a reference, more effective than paclitaxel. SorC27 was also, in all cases, more effective in inducing a faster and more intense apoptotic cascade than paclitaxel. Due to recent findings that calcium flow through TRPV6 can initiate an antiapoptotic response in cancer cells, which is communicated through the NFAT transcription factor circuit (Lehen'kyi et al., 2007) and that reducing the amount of TRPV6 in the breast cancer T 47D cell line enhances the effect of

10 Tamoxifen, a taxol (Bolanz et al., 2008), the co-treatment of any of the C peptides (or a combination of them) with any of the taxols would lead to an enhancement of cancer treatment.

Figure 4 illustrates the comparison of SorC13 and paclitaxel in the T 47D human breast cancer cell line and the comparison of SorC27 and paclitaxel in the MCF7 human breast cancer cell line. Apoptosis was induced more rapidly by SorC peptides in these two cell lines, and the apoptotic cascade was more

15 intense. Similar effects of an earlier and more intense onset of the apoptotic cascade were observed in MB 468 and MB231 by means of the two SorC peptides.

Example 5: Analysis of SorC13 and SorC27 with paclitaxel in enhancing the induction of apoptosis in the metastatic human breast cancer cell lines

To evaluate the effect of both type C peptides by comparison with a reference treatment currently

20 used against breast cancer (paclitaxel), the combinations of SorC13 (10 and 100 µM), SorC27 (10 and 100 µM) and paclitaxel (10 µM) are compared in the human breast cancer cell lines MCF7 (of the ATCC HTB-22 and T 47D (from the ATCC HTB-133) Cell lines are prepared according to ATCC procedures, both the APO-ONE (Promega) and the CellTiter Blue assays are used with the cell line panel of breast cancer The treatment groups are as follows:

25 (n = 8 per treatment)

1) Without treatment (control)

2) Treat with paclitaxel (10 µM)

3) Treat with 2 doses of SorC13 (10, 100 µM)

4) Treat with 2 doses of SorC27 (10, 100 µM)

30 5) Treat with a single dose of paclitaxel (10 µM) and 2 doses of SorC13 (10, 100 µM)

6) Treat with a single dose of paclitaxel (10 µM) and 2 doses of SorC27 (10, 100 µM)

Data are compared directly in terms of average response and analyzed for a significant difference.

SorC27 or SorC13 polytherapy with paclitaxel has a greater induction effect of apoptosis in the lines

35 breast cancer cell phones than any of the monotherapeutic treatments. These results are measured as an apoptotic index (AI = response to treatment / response to control; AI = 1 when there is no effect) and it is determined whether cell viability decreased (+) or not (-).

Example 6: Analysis of SorC13 and SorC27 against the paclitaxel / carboplatin cocktail when inducing apoptosis in human ovarian cancer cell lines

40 To compare the effect of the two SorC peptides against the reference treatment for ovarian cancers, SorC13 or SorC27 was compared with the treatment using a carboplatin and taxol (CAT) cocktail (10 µM paclitaxel, 20 nM carboplatin ) with the APO-ONE trial, which measures the combined activities of caspase 3 and caspase 7. Due to the mixed treatment (CAT), the effects were compared in terms of the proportion of treatment: control. If no effect was noted, then the ratio was equal to 1. The comparisons of the

45 treatment proportions: control were examined with the Student t test with a 95% confidence interval. The proportions larger than a unit value indicated a positive effect. The effects are illustrated in Figure 5. Figure 6 shows a concentration effect of SorC27 on one of the ovarian cell lines. These figures illustrate that:

 SorC27 (10 µM and 100 µM) caused earlier and more intense apoptosis than the CAT cocktail in 50 the SKOV3 and OVCAR3, and was similar to the CAT in CaOV3 and OV90.

 SorC13 produced an earlier and more intense apoptotic cascade than the CAT cocktail for SKOV3, CaOV3, OVCa3 and OV90.

Example 7: Analysis of SorC13 or SorC27 with the paclitaxel / carboplatin cocktail when enhancing the induction of apoptosis in the metastatic human ovarian cancer cell lines

5 To compare the effect of the two C-peptides against the reference treatment for ovarian cancers, SorC13 and SorC27 are compared with the treatment using a carboplatin and taxol (CAT) cocktail (paclitaxel at 10 µM, carboplatin at 20 µM) with the APO-ONE trial. Two human ovarian adenocarcinoma tumor cell lines, SKOV3 (from ATCC HTB-77) and NIH: OVCAR-3 (from ATCC HTB-161), are used and prepared according to the ATCC procedures. Due to the mixed treatment (CAT), the effects are compared in terms of the proportion of

10 treatment: control. If no effect is noted, then the ratio is equal to 1. Comparisons of the treatment: control ratios are examined. The proportions above one indicate a positive effect. The treatment groups are as follows:

(n = 8 per treatment)

1) Without treatment (control)

15 2) Treat with paclitaxel (10 µM)

3) Treat with 2 doses of SorC13 (10, 100 µM)

4) Treat with 2 doses of SorC27 (10, 100 µM)

5) Treat with a single dose of paclitaxel (10 µM) and 2 doses of SorC13 (10, 100 µM)

6) Treat with a single dose of paclitaxel (10 µM) and 2 doses of SorC27 (10, 100 µM)

20 Data are compared directly in terms of average response. The effects of the combination of SorC27 + paclitaxel and SorC13 + paclitaxel on one of the ovarian cell lines show that:

 SorC27 (10 µM and 100 µM) in polytherapy with the CAT cocktail enhances apoptosis sooner and with more intensity than any monotherapeutic treatment in SKOV3 and NIH ovarian cancer cell lines: OVCAR-3.

25  SorC13 (10 µM and 100 µM) in polytherapy with the CAT cocktail enhances apoptosis sooner and with more intensity than any monotherapeutic treatment in the SKOV3 and NIH ovarian cancer cell lines: OVCAR-3.

These results are measured as an apoptotic index (AI = response to treatment / response to control; AI = 1 when there is no effect) and determining whether cell viability decreases (+) or not (-).

30 Example 8: Tissue distribution of SorC13 and SorC27

SorC13 and SorC27 were labeled with the near infrared probe Cy5.5. SorC13 was labeled in lysine 1 and lysine 8 with the cy5.5 infrared fluorescent probe through the reaction with the NHS ester activated procedure of Cy5.5. SorC27 was labeled in its only cysteine thiol with the maleamide activated reaction of Cy5.5. The labeled peptides were purified with a combination of size exclusion chromatography and HPLC. The marking, Cy5.5, emits fluorescence in the infrared region after excitation with a scanning laser. The low energy laser is able to penetrate the animal up to about 1 cm and in this way, by scanning in the prone and supine positions, the presence of labeled peptides can be quantified in three dimensions.

Cy5.5-labeled peptides were injected intravenously into CD1 mice (4 for each compound) at 100 µg per animal in 100 µl, and the animals were radiographed alive with an optical imaging system, 40 Optix eXplorer ( GE Healthcare Systems) at different points of time (30 min, 90 min, 4 h). Some animals were observed 24 hours after infusion to remove blood (and lymph). The biodistribution of the labeled peptides in different organs and tissues were visualized and relatively quantified by analysis with optical imaging. This protocol allows to visualize the location of the labeled peptides and how the location changes over time. Figure 7 shows the location of the lymph nodes in the mouse. The

45 nodes that accumulated labeled peptides are indicated by line 1 (superfacial cervical nodes), line 4 (axillary nodes), line 5 (gill nodes), line 8 (mesenteric nodes) and line 9 (inguinal nodes) . Figures 8, 9 and 10 show the amount of labeled peptides in different organs ex vivo. The set, these experiments show that:

 None of the C peptides crossed the blood brain barrier

 Marked SorC13 and SorC27 are predominantly located in the lymph nodes, lung, liver and kidney.

 SorC13 and SorC27 brands were still detectable in these tissues after infusion at 24 hours.

5  The measurement of the shelf life of fluorescence in the different organs showed that the metabolism of the labeled peptides appears in the liver and kidney, since Cy5.5 has a shorter shelf life than the peptide adducts / Cy5.5 .

Example 9: SorC13 and SorC27 induce apoptosis in human non-small cell lung carcinoma (NSCLC) tumor cells

10 The effects of C-peptides on human lung carcinoma cells are studied. The tissue distribution data described above indicated that C peptides accumulate in the lung and are located in the lymph nodes. In vitro studies using SorC13 and SorC27 are carried out on cell lines from metastatic lymph nodes to determine their effect on human non-small cell lung carcinoma (NSCLC) cell lines H1437 (from ATCC CRL-5872) and H2087 (from the ATCC CRL

15 5922). Both cell lines are adenocarcinomas from the metastatic site (lymph node) and stage 1 NSCLC lung cancer. The main lymph nodes of interest that are usefully treated with the peptides of the invention include the superfacial cervical nodes, the axillary nodes, the gill nodes, mesenteric nodes and inguinal nodes.

For the study, cell lines are grown under recommended conditions and in the culture medium

20 recommended by the ATCC. Cell lines are grown under sterile conditions at 37 ° C, in a humidified atmosphere of 5% CO2.

Both the APO-ONE assay and the CellTiter Blue (Promega) are used in cell lines to assess apoptosis and cell viability, respectively. The treatment groups are as follows:

1) Without treatment

25 2) Treat with 2 doses of SorC13 (10 and 100 µM)

3) Treat with 2 doses of SorC27 (10 and 100 µM)

Data are compared directly in terms of average response. SorC27 and SorC13 induce apoptosis in human metastatic NSCLC lines.

Examples 10-13: The effects of C peptides are evaluated in a series of human cancer cell lines

30 The effects of treatment with SorC13 and SorC27 on a series of cancer cell lines are analyzed in vitro. As described above, C-peptides induced apoptosis in breast cancer cell lines and in ovarian cancer. C peptides are also capable of inducing apoptosis and decreasing cell viability in other cancer cells in vitro. The following examples 10-13 assess the ability of SorC13 and SorC27 to induce apoptosis in a selected set of human cancer lines. They include: PC3,

35 human chronic myelogenous leukemia (CML), K-562, human acute myelogenous leukemia (AML), MV-4-11, Daudi cell line of Burkitt lymphoma.

For this work, the concentrations of SorC3 and SorC27 used are 0 µM, 10 µM and 100 µM. The experiments extend over a period of 5 to 7 days. All measurements are made in quadruplicate and to correct the test measurements a blank (without cells) and a control (vehicle, without peptides) were used. The data from

The response as a function of time and the response data as a function of the dose are plotted as means ± the standard error of the mean.

Example 10: C-peptides induce apoptosis in human prostate cancer cell lines

The PC3 cell lines (from the ATCC CRL-1435) and the FGC clone from LnCaP (from the ATCC CRL-1740), were obtained from the American Type Culture Collection (ATCC) and were grown under the recommended conditions and in the

45 culture media recommended by the ATCC. Cell lines are grown under sterile conditions at 37 ° C, in a humidified atmosphere of 5% CO2.

The induction of apoptosis and the accompanying decrease in cell viability in cancer cell lines are observed. From the large increases in the activities of caspases 3-7, it is clear that the induction of apoptosis depends on a time-dependent effect and a dose-dependent effect.

50 Observations of the effects of SorC13 and SorC27 on the cell line are:

 SorC27 induces apoptosis and decreases cell viability;

 SorC13 induces apoptosis and decreases cell viability. Example 11: C-peptides induce apoptosis in human Burkitt lymphoma cell lines. The Daudi cell line (from ATCC CCL-213) is grown under the recommended conditions and in the media.

5 crops recommended by the ATCC. Cell lines were cultured under sterile conditions at 37 ° C, in a

humidified atmosphere of 5% CO2. The induction of apoptosis and the accompanying decrease in cell viability in cell lines are observed. Of cancer. From the large increases in the activities of caspases 3-7, it is clear that the induction of Apoptosis depends on a time-dependent effect and a dose-dependent effect.

10 Observations of the effects of SorC13 and SorC27 on the cell line are:  SorC27 induces apoptosis and decreases cell viability.  SorC13 induces apoptosis and decreases cell viability.

Example 12: C-peptides induce apoptosis in the human myelogenous leukemia (LMC) cell line, K-562

15 The K-562 cell line (from the ATCC CCL-243) is grown under the recommended conditions and in the culture media recommended by the ATCC. Cell lines were grown under sterile conditions at 37 ° C, in a humidified atmosphere of 5% CO2.

The induction of apoptosis and the accompanying decrease in cell viability in cancer cell lines are observed. From the large increases in the activities of caspases 3-7, it is clear that the induction of apoptosis depends on a time-dependent effect and a dose-dependent effect. The effects of SorC13 and SorC27 on the cell line are:  SorC27 induces apoptosis and decreases cell viability;  SorC13 induces apoptosis and decreases cell viability. Example 13: C-peptides induce apoptosis in the human acute myelogenous leukemia (AML) cell line,

25 MV-4-11 The MV-4-11 cell line (from the ATCC DRL-9591) is grown under the recommended conditions and in the culture media recommended by the ATCC. Cell lines were grown under sterile conditions at 37 ° C, in a humidified atmosphere of 5% CO2.

The induction of apoptosis and the accompanying decrease in cell viability in cancer cell lines is observed. From the large increases in the activities of caspases 3-7, it is clear that the induction of apoptosis depends on a time-dependent effect and a dose-dependent effect. The observations of the effects of SorC13 and SorC27 on the cell line are:  SorC27 induces apoptosis and decreases cell viability;  SorC13 induces apoptosis and decreases cell viability.

35 Examples 14-20: SorC peptides have antitumor activity in vivo In vivo studies (examples 14-20) that employ C peptides in a xenograft model are carried out to evaluate the response to treatment of xenografts of Human cancer cell lines in a rodent model (mouse). SorC peptides have antitumor activity in vivo.

The cell lines used as a model of human breast cancer and human ovarian cancer are

40 obtained from the American Type Culture Collection (ATCC), and grown under the recommended conditions and in the culture media recommended by the ATCC. The cell types, the ATCC catalog number and the description of the cell line are shown in Table 8.

Table 8: Description of the ATCC cell lines

NIH-OVCAR-3

The NIH: OVCAR-3 line was established in 1982 by T. C. Hamilton et al. from malignant ascites of a patient with a progressive ovarian adenocarcinoma.

(ATCC HTB-161)

The cell line is resistant to clinically relevant concentrations of adriamycin, melphalan and cisplatin. Xenograft models have been used to demonstrate that treatment with 17-βestradiol induces progesterone receptors in this human ovarian carcinoma. NIH: OVCAR-3 is an appropriate model system in which to study pharmacological resistance in ovarian cancer, and the presence of hormonal receptors is useful for evaluating hormonal treatment (of the ATCC HTB-161). TRPV6 mRNA is expressed (the trpv6 genes are on) in this ovary cell line.

SKOV3

SKOV3 cells of malignant ascites of a patient with a progressive ovarian adenocarcinoma.

(ATCC HTB-77)

This cell line is resistant to tumor necrosis factor and several cytotoxic drugs, including diphtheria toxin, cisplatin and adriamycin (from the ATCC HTB-77). TRPV6 mRNA is expressed (the trpv6 genes are on) in this ovary cell line.

Daudi: Burkitt lymphoma (ATCC CCL-213)

The Daudi line was obtained by E. Klein and G. Klein in May 1967 from a 16-year-old man with Burkitt lymphoma. Cells do not express β2 microglobulin. They do express EBNA, VCA and surface immunoglobulin (sIg +). Daudi is a well-characterized lymphoblast B cell line that has been extensively used in studies of the mechanisms of leukemia (from ATCC CCL213).

Human chronic myelogenous leukemia (CML) cell line, K-562 (ATCC CCL-243)

The continuous cell line K-562 was established by Lozzio and Lozzio from the pleural effusion of a 53-year-old woman with chronic myelogenous leukemia in terminal hemoblastic crisis [PubMed: 163658]. The studies conducted by Anderson et al. on the membrane surface properties they showed that K-562 was a human erythroleukemia line. The cell line is oncogenic in nude mice (tumors developed in less than 21 days with a frequency of 100% (5/5) in nude mice inoculated subcutaneously with 107 cells) (from the ATCC CCL-243) .

Human acute myelogenous leukemia (AML) cell line, MV-4-11 (ATCC CRL-9591)

The MV4-11 cell line was established by Rovera and collaborators from the blasts of a 10-year-old male with biphenotypic B-myelomonocytic leukemia. This line was first called ATCC HTB-189 (from the ATCC CRL-9591).

Clone FGC of LnCaP, human prostate cancer (ATCC CRL-1740)

The FGC clone of LnCaP was isolated in 1977 by JS Horoszewicz et al., From a needle-suction biopsy of the left supraclavicular lymph node of a 50-year-old white male (type B + blood) with a confirmed diagnosis of carcinoma of metastatic prostate. They adhere only slightly to the substrate, do not become confluent and quickly acidify the medium. Growth is very slow (from the ATCC CRL-1740).

PC3 cell line of

PC-3 started from a bone metastasis of a prostate adenocarcinoma of

adenocarcinoma of

grade IV of a 62 year old white male.

human prostate

Tumors developed in less than 21 days with a frequency of 100% (5/5) in

(ATCC CRL-1435)

athymic mice inoculated subcutaneously with 10 (7) cells. The cells show little activity of acid phosphatase and testosterone-5-α reductase (from ATCC CRL-1435).

MCF7 human breast cancer cell line (ATCC HTB-22)

It comes from a metastatic site such as a pleural effusion. The primary tumor came from the breast in a patient with an epithelial adenocarcinoma. The MCF7 line retains several characteristics of the differentiated mammary epithelium, including the ability to process estradiol through cytoplasmic estrogen receptors and the ability to form domes. The cells express the WNT7B oncogene [PubMed: 8168088]. It contains the Tx-4 oncogene. The growth of MCF7 cells is inhibited by tumor necrosis factor α (TNF α). IGFBP secretion can be modulated by treatment with antiestrogens (ATCC # HTB-22).

MDAMB-231 human breast cancer cell line (ATCC HTB-26)

It comes from a metastatic site such as a pleural effusion. The primary tumor came from the breast of a patient with epithelial adenocarcinoma. The cells express the WNT7B oncogene. MDA-MB-231 cells express a small amount of HER2 (from the ATCC HTB-26).

Example 14: SorC13 and SorC27 antitumor activity in monotherapy and in polytherapy with CAT (carboplatin and paclitaxel cocktail) against human ovarian adenocarcinoma tumor cells in a mouse xenograft model

5 The human ovarian adenocarcinoma cell lines, SKOV3 (ATCC # HTB-77) and NIH: OVCAR-3 (ATCC # HTB-161) are grown in the growth media prepared with the complete growth medium of the ATCC.

Cell cultures

SKOV3 cell line

The base medium for the SKOV3 line is the modified McCoy medium 5a formulated by the ATCC (No. 30-2007 of the

10 ATCC catalog). To make the complete growth medium, the following components are added to the base medium: fetal bovine serum at a final concentration of 10% at 37 ° C (air, 95%; carbon dioxide (CO2), 5%). The ATCC subculture protocol is followed for SKOV3 cell cultures.

NIH-OVCAR-3 cell line

The base medium for the NIH-OVCAR-3 cell line is the RPMI 1640 medium formulated by the ATCC (No. 30-2001

15 of the ATCC catalog). The ATCC propagation protocol for NIH-OVCAR-3 cell cultures is followed to make the growth medium complete.

Cell preparation

Cells that have been cryopreserved in liquid nitrogen are rapidly thawed at 37 ° C and transferred to a tissue culture flask containing the growth medium and then incubated at 37 ° C in an incubator

20 with 5% CO2. To propagate the cell line, cultures are passed 1: 2 at a density of 5 x 10 6 cells / ml every three days by adding an equal volume of freshly prepared growth medium. When the bottles reach a density of approximately 10 x 10 6 cells / ml, the above pass procedure is repeated until sufficient cells are obtained for implantation in the mice.

From 4 to 5 NOD / SCID mice from seven to eight weeks of age were housed in a cage in micro-insulators, with a light / dark cycle of 12 h / 12 h, acclimatized for at least 1 week before using them and fed with

I think of normal laboratory at will. Studies were carried out with animals that were between 8 and 12 weeks old at the time of implantation of the tumor cells.

Formulation procedures

SorC13 / SorC27

5 To formulate SorC13 and SorC27, the stock solutions of the test article are prepared by dissolving the appropriate amounts of the compound in physiologically buffered saline (pH 7.0 to 7.4 at 25 ° C) to comprise a final solution with which to administer 10 and 100 mg / kg of mouse. Stock solutions are prepared weekly, stored at –20 ° C and diluted new every day for dosing. All solutions are sterilized by filtration with a 0.22 µm filter before further handling. The units sterilized by

The filtration was previously washed with a small volume of sterile distilled water.

Carboplatin / paclitaxel reference solution (CAT)

The stock solutions are prepared with 20 mM and 10 µM carboplatin and paclitaxel, respectively. Carboplatin and paclitaxel are obtained from Sigma-Aldrich. All solutions are sterilized by filtration with a 0.22 µm filter before further handling.

15 Implementation Procedures

Optionally, to implant the NIH-OVCAR-3 or SKOV3 tumor cells in the NOD / SCID mice, the cell cultures are centrifuged to sediment the cells, the supernatant is aspirated, the cell pellet is resuspended in 10 ml of the growth medium and Determine the number of cells with a hemocytometer. The cells are then washed in the appropriate medium and resuspended at a concentration of 5 x 10 7 cells / ml in the medium.

20 With a 27 gauge needle and a 1 cm3 syringe, 0.1 ml of the cell suspension was injected subcutaneously into the sides of the mice. The tumors are then allowed to develop in vivo until the majority reaches a tumor volume of 100 to 200 mm 3, which typically takes 1 to 2 weeks after implantation. These tumors are used to produce subcutaneous tissue implants in NOD / SCID mice that, after 2 days, are treated with the problem peptides or with CAT.

25 The following groups are typically included:

1) Mice with tumor implants that receive the vehicle;

2) Mice with tumor implants receiving the CAT reference compound;

3) Mice with tumor implants receiving dose 1 of SorC13;

4) Mice with tumor implants receiving dose 1 of SorC27;

30 5) Mice with tumor implants receiving dose 1 of SorC13 and CAT;

6) Mice with tumor implants receiving dose 1 of SorC27 and CAT. GONZ

Measurements

Animals with oblong tumors, very small or large, are discarded, and only tumor-bearing animals showing a repetitive growth rate are selected for studies. The volume of each tumor (V)

35 is calculated by measuring with calipers using standard measurements of the width (A), height (H) and length (L) of the tumors. The animals are distributed randomly in the treatment groups, so the median tumor volume of each group was similar at the beginning of the dosage. The percentage of T / C values is determined as a measure of efficacy, where C refers to the volume of the untreated or control tumor, and T refers to the volume of the treated tumor.

40 Treatment Procedures

Animals are injected intraperitoneally (i.p.) this formulation to 10 ml / kg of body mass in one dosage.

Results

Treatment with a dose of 400 mg / kg of body mass of SorC13 and SorC27 substantially decreases the

Growth rate of NIH-OVCAR-3 and SKOV3 cells in NOD / SCID mice. This effect is not associated with toxicity, as evidenced by the stability of the body mass and the absence of symptoms of stress (diarrhea, gasps, bristly fur).

Experimental results with SKOV3 and SorC27 cells

SKOV3 cells were obtained from the ATCC, cultured and expanded to an adequate amount to establish xenografts in mice, and then formulated in gel granules and grafted under the suprarenal capsule of replacement NOD / SCID mice to establish tumors Then you let the

5 tumors develop in vivo until most reach 100-200 mm3 of tumor volume, which typically occurs within 1 to 2 weeks after implantation. Once the xenografts have been established, they are collected and cut into many uniform pieces. For experimentation, sixteen to eighteen pieces were implanted below the skin in each NOD / SCID mouse. After 2 days, the animals are treated with the problem peptides either with CAT or polytherapies.

10 Mice receiving implants with SKOV3 cells as detailed above received saline (control), SorC27 (400 mg / kg), by injection i.p. every day for 12 days, or CAT injections once a week. Each day, tumor volume and body mass were measured. Likewise, the general health of the animals was checked every day. The results shown in Figure 12 are presented in terms of tumor load normalized by body mass. Both treatments with SorC27 and CAT showed a huge reduction in tumor volume

15 and were significantly different from the control. The percentage of the T / C ratio (mean tumor volume of treaties / mean tumor volume of controls) was 39.9%, reflecting a 60.1% decrease in tumor growth. By day 12 there was no statistically significant difference between CAT and SorC27 with this dose.

Example 15: SorC13 and SorC27 antitumor activity in monotherapy and in paclitaxel polytherapy against the 20 human breast cancer cells in a mouse xenograft model

The human breast cancer cell lines, MCF7 (ATCC # HTB-22) and T 47D (ATCC # HTB-133) are grown in the growth media prepared with the complete growth medium of the ATCC.

Cell cultures, cell preparation, animal procedures, SorC13 / SorC27 formulation procedures, implantation procedures, measurements and treatment procedures are as described in example 14.

The following groups are typically included:

one)

Mice with tumor implants that receive the vehicle;

2)

Mice with tumor implants receiving paclitaxel, the reference compound;

3)

Mice with tumor implants receiving dose 1 of SorC13;

30

4) Mice with tumor implants receiving dose 1 of SorC27;

5)

Mice with tumor implants receiving dose 1 of SorC13 and paclitaxel;

6)

Mice with tumor implants receiving dose 1 of SorC27 and paclitaxel.

Formulation procedures Reference solution with paclitaxel

35 Stock solutions containing 10 µM paclitaxel are prepared. Paclitaxel is obtained from Sigma-Aldrich. Results Treatment with a dose of SorC13 or SorC27, optionally from 200 to 400 mg / kg of body mass, decreases

substantially the growth rate of MCF7 and T 47D cells in NOD / SCID mice, with a percentage of the T / C value of less than 100%. This effect is not associated with toxicity, as evidenced by the reduced

40 percentage of T / C throughout the development of the study. Example 16: SorC13 and SorC27 antitumor activity against human chronic myelogenous leukemia (LMC) tumor cells, [K-562 (ATCC # CCL-243)], in a mouse xenograft model

The human chronic myelogenous leukemia (CML) cell line, K-562 (ATCC # CCL-243), is obtained from the American Type Culture Collection. The cells are grown in the growth media prepared with the complete growth medium of the ATCC. Cell cultures The base medium for the K-562 line is Iscove's modified Dulbecco medium formulated by ATCC, No. 30

2005 of the catalog. To manufacture the complete growth medium, the following components are added to the base medium: fetal bovine serum at a final concentration of 10%. To manufacture the complete growth medium, the ATCC propagation protocol for K-562 cell cultures is followed.

The following groups are typically included:

5 1) Mice with tumor implants that receive the vehicle; 2) Mice with tumor implants receiving dose 1 of SorC13; 3) Mice with tumor implants receiving dose 1 of SorC27;

Cell preparation, animal procedures, SorC13 / SorC27 formulation procedures, implantation procedures, measurements and treatment procedures are as described in Example 14.

Results Treatment with a dose of SorC13 or SorC27, optionally from 200 to 400 mg / kg of body mass, substantially decreases the growth rate of K-562 cells in NOD / SCID mice, with a percentage of the value of T / C of less than 100. This effect is not associated with toxicity.

15 Example 17: SorC13 and SorC27 antitumor activity against human prostate cancer tumor cells,

LnCaP FGC clone, in a mouse xenograft model The human prostate cancer cell line, LnCaP FGC clone (from the ATCC CRL-1740), is obtained from the American Type Culture Collection. The cells are grown in the growth media prepared with the complete growth medium of the ATCC.

20 Cell cultures The base medium for the LnCaP FGC clone line is formulated according to the ATCC. Procedures with animals As described in example 14 The following groups are typically included:

25 1) Mice with tumor implants receiving the vehicle; 2) Mice with tumor implants receiving dose 1 of SorC13; 3) Mice with tumor implants receiving dose 1 of SorC27.

Cell preparation, SorC13 / SorC27 formulation procedures, implantation procedures, measurements and treatment procedures are as described in example 14.

30 Results Treatment with a dose of SorC13 or SorC27, optionally from 200 to 400 mg / kg of body mass, substantially decreases the growth rate of LnCaP cells in SCID mice, with a percentage of the T / C value of less than 100. This effect is not associated with toxicity, as evidenced by the reduced percentage of T / C throughout the study.

35 Example 18: SorC13 and SorC27 antitumor activity against human breast cancer tumor cells,

MCF7, in a mouse xenograft model The human breast cancer cell line, MCF-7 (from the ATCC HTB-22), is obtained from the American Type Culture Collection. The cells are grown in the growth media prepared with the complete growth medium of the ATCC.

40 Procedures with animals As described in Example 14 The following groups are typically included:

1) Mice with tumor implants that receive the vehicle; 28

2) Mice with tumor implants receiving dose 1 of SorC13;

3) Mice with tumor implants receiving dose 1 of SorC27;

Cell culture, cell preparation, SorC13 / SorC27 formulation procedures, implantation procedures, measurements and treatment procedures are as described in Example 14.

Results

Treatment with a dose of SorC13 or SorC27, optionally from 200 to 400 mg / kg of body mass, substantially decreases the growth rate of MCF7 cells in NOD / SCID mice, with a percentage of the T / C value of less of 100. This effect is not associated with toxicity, as evidenced by the reduced percentage of

10 T / C throughout the development of the study.

Example 19: SorC13 and SorC27 antitumor activity against human breast cancer tumor cells, MDA-MB-231, in a mouse xenograft model

The human breast cancer cell line, MDA-MB-231 (from the ATCC HTB-26), is obtained from the American Type

Culture Collection The cells are grown in the growth media prepared with the complete growth medium of the ATCC.

Cell cultures

The base medium for the MDA-MB-231 cell line is Leibovitz L-15 medium formulated by ATCC, No. 302008 in the catalog. To manufacture the complete growth medium, the following components are added to the base medium: fetal bovine serum at a final concentration of 10%. To manufacture the complete growth medium, it is

20 follows the ATCC propagation protocol for MDA-MB-231 cell cultures.

Procedures with animals

As described in example 14

The following groups are typically included:

one)

Mice with tumor implants that receive the vehicle;

25

2) Mice with tumor implants receiving dose 1 of SorC13;

3)

Mice with tumor implants receiving dose 1 of SorC27;

Cell preparation, SorC13 / SorC27 formulation procedures, implantation procedures, measurements and treatment procedures are as described in example 14.

Results

30 Treatment with a dose of SorC13 or SorC27, optionally from 200 to 400 mg / kg of body mass, substantially decreases the growth rate of MDA-MB-231 cells in SCID mice, with a percentage of the value of T / C of less than 100. This effect is not associated with toxicity, as evidenced by the reduced percentage of T / C throughout the study.

Example 20: SorC13 and SorC27 antitumor activity against human acute myelogenous leukemia 35 (AML) tumor cells, MV-4-11, in a mouse xenograft model

The human breast cancer cell line, MV-4-11 (from the ATCC CRL-9591), is obtained from the American Type Culture Collection. The cells are grown in the growth media prepared with the complete growth medium of the ATCC.

Cell cultures

40 The base medium for the MV-4-11 cell line is Iscove's modified Dulbecco medium formulated according to ATCC, No. 30-2005 of the catalog. To manufacture the complete growth medium, the following components are added to the base medium: fetal bovine serum at a final concentration of 10%. The ATCC propagation protocol for MV-4-11 cell cultures is followed to manufacture the complete growth medium.

Procedures with animals As described in example 14. The following groups are typically included:

one)

Mice with tumor implants that receive the vehicle;

5

2) Mice with tumor implants receiving dose 1 of SorC13;

3)

Mice with tumor implants receiving dose 1 of SorC27;

The preparation of the cells, the formulation procedures of SorC13 / SorC27, the procedures of

Implantation, measurements and treatment procedures are as described in example 14.

Results

10 Treatment with a dose of SorC13 or SorC27, optionally from 200 to 400 mg / kg of body mass, substantially decreases the growth rate of MV-4-11 cells in SCID mice, with a percentage of the value of T / C of less than 100. This effect is not associated with toxicity, as evidenced by the reduced percentage of T / C throughout the study.

Example 21: Detection and detection limits of C peptides

15 Reverse phase HPLC was used to detect and quantify SorC13 and SorC27. For the analytical procedures a Phenomenex Gemini column, 5 u, C-18, reverse phase (250 x 4.60 mm) was used. The solvent system was a gradient of acetonitrile and water, each with 0.1% v / v trifluoroacetic acid. HPLC was carried out at a column temperature of 30.0 ° C. The solvent system was a gradient of 10% to 60% acetonitrile (0.1% TFA) with 90% to 40% water (0.1% TFA) for 40 minutes, at a flow rate of 1 , 0 ml / min. The

20 detection was at 224 nm. The retention time of the peptides was 11.5 min and 14.7 min for SorC13 and SorC27, respectively.

The quantification and detection limits of SorC13 and SorC27 in the water were determined by diluting

sequentially solutions containing known masses of pure peptides and subject to analysis by

HPLC In the case of quantifying the two C peptides in rat or human plasma, the procedure required

25 pretreatment that eliminates proteolytic activity of plasma samples. A combination of the addition of 2 µl of Pefabloc (Sigma-Aldrich; a protease inhibitor) to 2 mM of Pefabloc per 100 µl of solution and centrifugation in an Amicon YM-10 Centricon size exclusion filter (MWCO 10 kDa) to remove proteolytic enzymes it provided a means of peptide stability. All quantification experiments were performed in triplicate and analyzed by calculating the best equation of the line in the 95% confidence interval.

30 The standard curves of the three media were adjusted to a linear model within the 95% (or greater) confidence interval. The lower detection limit of SorC13 and SorC27 in all media under these conditions was at least 1 µg in a sample injection volume of 94 µl. The decrease in detection limits for these peptides is easily achieved with preanalytical bypass for colorimetric and fluorimetric procedures. Additionally, decreasing the detection wavelength (e.g., 204 nm) also lowers the limit of

35 detection of peptides.

Example 22: Degradation rate of C-peptides in rat and human plasma

Rat plasma was obtained from blood taken from slaughtered rats (Sprague-Dawley) and placed in vials of

Heparinized glass to prevent clotting. Whole blood was centrifuged at 8000xg for 5 min to

sediment the cells and leave the plasma in the supernatant. Human whole blood samples from humans

40 healthy ones were taken in heparinized and lithium vacuum tubes. The samples were placed in 2.0 ml microcentrifuge tubes and centrifuged at 8000 xg for 5 min. The plasma was carefully separated from the red blood cells and used immediately.

For the determination of the degradation rate of the C peptides, the HPLC protocol measured the

Amount of SorC13 or SorC27 in the dosed plasma samples, after some time. The tubes of

45 samples (500 µl) containing 2 µl of Pefabloc at 2 mM were prepared beforehand for sample administration. SorC13 (1.0 mg) or SorC27 (2.0 mg) was dissolved in 1.0 ml of freshly prepared plasma (37 ° C) and placed in a water bath to incubate at 37 ° C. A sample (100 µl) was immediately taken and stopped in sample tubes with 2 µl of 2 mM Pefabloc, mixed vigorously and immediately frozen at –80 ° C. This initial sample represented the zero time of the experiment. At successive time intervals of 5 minutes,

50 samples were taken at 100 µl, stopped in Pefabloc, mixed and frozen at –80 ° C until analysis.

The degradation rate of rat plasma was determined for the plasma of three different rats, while

Six different plasma samples were used for the degradation rate of human plasma. The data used were the means (± SEM) and were adjusted to a simple exponential decomposition model of a phase to determine the half-life of the two peptides. This also allowed to calculate a static degradation rate. Figure 11 illustrates the rate at which SorC27 degraded in human plasma. The analysis

5 similar for the two peptides in both types of plasma showed:

 SorC27 degraded with a half-life of 21 min in rat plasma and 20 min in human plasma.

 SorC13 degraded with a half-life of 58 min in rat plasma and 56.6 min in human plasma.

Example 23: SorC13 and SorC27 toxicity studies

Acute toxicity of SorC13 and SorC27

10 As described below, a study of the toxicity by injection of an acute intravenous infusion in a single dose followed by a 5-day observation period in CD-1 mice. Three doses of each of SorC13 and SorC27 (10, 100 and 500 mg / kg) were used. Daily scans were performed next to the cage for 5 days with scans every hour for the first 4 hours after injection. As part of the analysis, the following were assessed: body mass before dosing and at the end; an autopsy of all animals was performed

15 (which includes a bone marrow smear preparation); the organs of all animals were weighed.

After injection i.v. In vivo in CD1 mice, SorC27 did not cause any change in blood pressure or heart rate during a measurement period of 1 hour. There were also no neurological / behavioral changes for 72 hours. After the injection i.v. in vivo in CD1 mice, SorC13 caused a small peak (approximately 25%) of blood pressure in the first 15 min, which disappeared after 1 hour. Do not

20 there were neurological / behavioral changes in the mice for 72 hours. The single intravenous injection of SorC13 or SorC27 in CD1 mice at doses of 10 mg / kg, 100 mg / kg and 500 mg / kg did not give rise to any adverse effects during an observation period of 5 days after injection. In this last experiment, the autopsy showed no significant change in any major organ system. Similarly, a multiple dose of SorC27 at 400 mg / kg (i.p.) every day for 12 days in the mice showed no indication of toxicity.

25 Study of the toxicity of repeated doses

To investigate the toxicity of repeated doses, SorC27 at 400 mg / kg formulated in saline solution was injected into NOD / SCID mice every day for 12 days. The animals were observed every day next to the cage and explored at the end of the experimental period. The comparison with control mice that were injected with saline alone did not show any toxic effects, weight loss or organ changes at autopsy.

30 Example 24: Pharmacokinetic (FC) profiles of SorC13 and SorC27

An FC study was performed by injection of acute intravenous infusion of a single dose using three doses of SorC13 and SorC27 (3 mg / kg, 30 mg / kg and 150 mg / kg) in at least 3 mice per dosage group. Blood samples were taken at 6 time points: 5, 15 minutes, 1, 2 and 4 h after the dose. Next, the presence of SorC13 or SorC27 was analyzed in the samples.

35 Results

The combination of the rate of elimination from the blood compartment plus enzymatic degradation due to plasma enzymes results in them disappearing from the blood compartment with a half-life of less than 30 minutes.

Example 25: Comparison of the presence of TRPV6 mRNA with the apoptotic activity of C peptides in 40 different cell lines

The presence of the TRPV6 mRNA was checked by RT-PCR in each of the cell lines collected in the

Table 9. TRPV6 primers (the size of the amplicon in an agarose gel is approximately 400 bp)

used were 5'- (direct primer) and 5 '

(reverse primer). The thermocycling program used for the amplification reaction was: 94 ° C for 3

45 minutes; It was then repeated for 30 cycles: 94 ° C for 30 seconds, 53 ° C for 30 seconds, 72 ° C for 1 min and 30 s; 72 ° C for 7 minutes. Amplification reactions were followed by standard agarose gel electrophoresis.

Results

Table 9 shows some correspondence between the ability of C-peptides to induce apoptosis in breast cancer and ovarian cancer cell lines, and the presence of TRPV6 mRNA. It is clear that cell lines expressing the trpv6 gene are susceptible to apoptosis induced by peptide C. 31

Table 9: Comparison of trpv6 gene expression and induction of apoptosis by SorC13 and Sorc27

Type of cancer

Cellphone line TRPV6 mRNA (RT-PCR) Apoptosis induced by C peptides

Mom

MB 231

(+) (+)

Mb 468

(+) (+)

T 47D

(+) (+)

HCC1954

(+) (+)

MCF7

(+) (+)

MCF 10A

(-) (-)

MCF 12A

(-) (-)

Ovary

OVCAR3

(+) (+)

OV C13

(+) Unanalyzed

SKOV-3

(+) (+)

OV 90

Unanalyzed (+)

OV 2008

(+) Unanalyzed

HEY C2

(+) (+)

Other SorC peptides of the invention, such as SorC9 (

), are easily analyzed with the protocols described in the previous examples and are shown to have in vitro and in vivo activity that inhibits calcium activity without any paralyzing activity. Peptides are also useful to prevent proliferation.

cell, induce apoptosis, and prevent or treat cancer.

Although the present invention has been described with reference to what is currently considered to be the preferred examples, it should be known that the invention is not limited to the examples described. On the contrary, the invention aims to cover the different modifications and equivalent preparations included within the scope of the

10 attached claims.

References

SEQUENCE LIST

<110> BioProspecting NB, Inc.

Stewart, John M. 5

<120> Peptide compositions for cancer treatment

<130> 15309-22 10 <150> US 61 / 037,903

<151>

<160> 2 15 <170> PatentIn version 3.3

<210> 1

<211> 27

<212> PRT 20 <213> Artificial sequence

<220>

<223> 25 <400> 1

<210> 2

<211> 18 30 <212> PRT

<213> Artificial sequence

<220>

<223> 35

<400> 2

Claims (16)

1. Isolated peptide comprising: a) an amino acid sequence with a sequence identity of at least 90% with the sequence of 5 b) at least 11 amino acids of the amino acid sequence: wherein said peptide inhibits calcium channel activity without paralyzing activity. 2. Peptide according to claim 1, comprising: a) amino acid sequence b) the amino acid sequence 10 c) the amino acid sequence 3. Peptide according to claim 1, consisting of: a) amino acid sequence b) the amino acid sequence c) amino acid sequence 4. Peptide according to any one of claims 1 to 3, wherein the calcium channel comprises a calcium channel of transient vanilloid-6 receptor potential (TRPV6). 5. Peptide according to any one of claims 1 to 4, wherein the peptide inhibits calcium uptake in a cancer cell.

6.

Peptide according to any one of claims 1 to 5, wherein the peptide induces apoptosis in a cancer cell.

7.

Peptide according to any of claims 1 to 6, to be used in treatments.

8.

Peptide according to any of claims 1 to 6, to be used in the treatment of cancer in an animal that needs it.

9.

Peptide for use in the treatment of cancer according to claim 8, wherein the cancer comprises breast cancer or ovarian cancer.

10. Peptide for use in the treatment of cancer according to claim 8, wherein the cancer comprises breast cancer, ovarian cancer, blood count, brain cancer, retinal cancer, liver cancer, thyroid cancer, cancer colon, colorectal cancer, prostate cancer, endometrial cancer or cervical cancer. 11. A peptide for use in the treatment of cancer according to any of claims 8 to 10, wherein the cancer comprises a metastatic cancer. 12. Peptide for use in the treatment of cancer according to claim 11, wherein the metastatic cancer is located in a lymph node, lung tissue, renal tissue, bone marrow or liver tissue. 13. Peptide for use in the treatment of cancer according to any of claims 8 to 12, 35 wherein the animal is a human.

14.

Pharmaceutical composition, comprising the peptide according to any one of claims 1 to 7 and a carrier.

fifteen.

Pharmaceutical composition according to claim 14, further comprising a second anti-cancer agent.

16. Pharmaceutical composition according to claim 15, wherein the second anticancer agent is a taxol.

17.

Pharmaceutical composition according to claim 15, wherein the second anticancer agent is tamoxifen.

18.

Procedure for inhibiting calcium uptake in a cancer cell in vitro, which comprises administering

to the cell the peptide according to any one of claims 1 to 6, wherein the peptide inhibits calcium uptake in the cancer cell. 19. Method for inducing apoptosis of a cancer cell in vitro, comprising administering to the cell the peptide according to any one of claims 1 to 6, wherein the peptide induces apoptosis in the cancer cell.